Sandra Almeida

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Bidirectional transcription of the repeat expansion generates sense and antisense RNAs that are translated into dipeptide repeat (DPR) proteins, but the mechanisms of translation initiation remain incompletely understood. Here, we used CRISPR-Cas9 genome editing and steric-blocking antisense oligonucleotides (ASOs) to investigate the role of AUG codons within the antisense repeat RNA. Deletion of an AUG-containing region upstream of the antisense repeats markedly reduced poly(GP) production without affecting antisense RNA levels, demonstrating that this sequence is required for efficient poly(GP) synthesis. We further found that unspliced sense transcripts containing the repeat expansion likely serve as templates for poly(GA) and poly(GR) production in motor neurons. Finally, ASOs targeting the antisense AUG-containing region reduced poly(PR) and poly(GP) levels without altering repeat RNA abundance, supporting a role for AUG-dependent translation of the antisense repeat RNA. These findings provide new insights into the mechanisms of DPR production and suggest that translation-blocking ASOs may represent a therapeutic strategy for C9ORF72-associated ALS/FTD.

The study by Chakraborty et al. investigates the functional relationship between two major PD-associated proteins, leucine-rich repeat kinase 2 (LRRK2) and α-synuclein (α-syn), during axonal transport. Using Drosophila as a model, the authors show that although LRRK2 does not significantly disrupt cargo movement, LRRK2 overexpression induces modest transport defects, suggesting a modulatory role rather than an essential role in transport regulation. Notably, specific PD-associated mutations in LRRK2 diXerentially influence αsyn-mediated transport impairments, revealing domain-specific eXects on vesicle dynamics. Strikingly, co-expression of LRRK2 alleviated α-syn-induced transport defects and reduced vesicle stalling independently of neuronal cell death, indicating that LRRK2 selectively modulates transport dynamics through mechanisms distinct from its kinasedependent activity. This study highlights the power of Drosophila genetics for resolving complex protein interactions underlying PD-related neuronal dysfunction.While the PD study focuses on intracellular transport dynamics, the focus shifts to toxic protein aggregation in AD in the study by De Plano et al., which explores the potential protective eXect of a bacteriophage identified through phage display technology against amyloid-beta (Aβ) toxicity. Aβ peptides accumulate in the brain and form amyloid plaques in AD, the most common neurodegenerative disorder. Among Aβ species, soluble oligomeric aggregates are considered particularly toxic, as they are strongly associated with synaptic dysfunction, oxidative and metabolic stress, and correlate more closely with cognitive decline than plaque burden. In this study, the bacteriophage 12CIII1 was shown to preferentially bind Aβ oligomers, with reduced aXinity for Aβ monomers and small oligomers, suggesting a capacity to selectively target toxic Aβ species. Consistent with this selectivity, preincubation with 12CIII1 protected diXerentiated SH-SY5Y neuroblastoma cells from Aβ42-induced neurite shortening, loss of cell viability, and transcriptional changes consistent with mitochondrial dysfunction and oxidative stress. Together, these findings support a role for metabolic impairment in AD-related neurodegeneration and identify 12CIII1 as a promising phage-based strategy to selectively target higher-order Aβ oligomeric assemblies.Complementing this focus on toxic protein species, Ham et al. expand the discussion toward neuroinflammation, a major pathological process increasingly recognized as a central driver of AD progression. The study leverages the Tg2576 transgenic mouse model that overexpresses a human amyloid precursor protein (APP) with the Swedish mutation, alongside Aβ-stimulated BV-2 murine microglial cells to probe neuroinflammation as a central driver of AD pathology. This model enables simultaneous evaluation of both amyloid burden and neuroinflammatory responses in a genetically defined context. This study demonstrates that Chitinase-3-like 1 (CHI3L1), a secreted glycoprotein which is upregulated in AD, could be a promising therapeutic target. A novel CHI3L1 monoclonal antibody, H1, was shown to cross the blood-brain barrier and, upon administration, alleviated memory impairment while reducing amyloid deposition. Mechanistically, H1 inhibited the extracellular signal-regulated kinase (ERK) and nuclear factor-kB (NF-κB) signaling pathways and suppressed pro-inflammatory microglial marker expression, results further confirmed by proteomic and gene expression analysis. Together, these findings position CHI3L1 neutralization as a promising immunotherapeutic strategy for attenuating neuroinflammation in AD.Broadening the perspective from protein aggregation and neuroinflammation to RNA metabolism and tissue vulnerability, the final contribution explores how stress-associated ribonucleoprotein assemblies may contribute to ALS pathogenesis. The mini-review by Ishaq and Russell provides a timely synthesis of how TDP-43-associated ribonucleoprotein assemblies may connect motor neuron degeneration with skeletal muscle pathology in ALS. This review bridges stress granules in neural tissues with myogranules in regenerating muscle, positioning these dynamic RNA-protein condensates not simply as bystanders of cellular stress but as potential nodes where impaired RNA metabolism, defective proteostasis, cytoplasmic TDP-43 accumulation, and tissue vulnerability converge. The authors emphasize that granule formation may be protective, compensatory, or pathological depending on context, duration, composition, and clearance, and avoid overstating liquidliquid phase separation as a universal causal mechanism. While the evidence directly linking myogranules to ALS remains elusive and largely correlative, this review highlights the importance of considering ALS pathogenesis through integrated neuron-muscle interactions rather than solely neuron-intrinsic mechanisms.The articles included in this Research Topic collectively employ diverse model systemsfrom Drosophila and transgenic mice to diXerentiated human neuronal cell lines, to investigate emerging mechanisms of neurodegeneration across molecular, cellular, and organismal scales. Together, these studies provide new insights into multiple aspects of pathogenesis, including domain-specific protein interactions in PD, amyloid aggregation toxicity and neuroinflammation in AD, and TDP-43-associated ribonucleoprotein assemblies in ALS. In addition, several contributions explore therapeutic strategies aimed at selectively interfering with toxic protein species and downstream pathogenic mechanisms. Importantly, this collection highlights how simplified and genetically tractable systems can reveal causal mechanisms that are often diXicult to resolve in complex mammalian disease contexts. As NDDs increasingly emerge as multifactorial diseases driven by interconnected pathways...

Neuromuscular disorders (NMDs) are a heterogeneous group of conditions that impair muscle and/or peripheral nerve function, leading to progressive weakness, sensory deficits, and muscle wasting. Although many NMDs are primarily driven by genetic mutations, their onset and progression are critically influenced by epigenetic mechanisms and environmental interactions, resulting in substantial phenotypic variability and heterogeneous disease trajectories. This genetic and phenotypic complexity underscores the urgent need to identify and develop robust biomarkers that can accelerate the transition from disease onset to molecular diagnosis and inform the assessment of therapeutic efficacy.The goal of this Research Topic is to showcase studies that not only explore novel biomarkers but also evaluate their utility in clinical settings, with the aim of enabling effective, personalized treatment strategies. By advancing our understanding of the molecular underpinnings of NMDs, this collection seeks to facilitate the translation of biomarker discovery into clinically meaningful, patient-specific therapies. Below, we summarize the nine articles included in this Research Topic, organized by disorder.Muscular dystrophies (MD) are a subset of NMDs driven by primary genetic defects that lead to progressive muscle degeneration. Despite extensive mechanistic insight, population level frameworks capable of identifying modifiable MD risk factors remain limited. Gong et al. address this gap by examining the association between the Neutrophil Percentage-to-Albumin Ratio (NPAR) and MD prevalence in 3,416 participants. Each unit increase in NPAR was associated with higher odds of MD with a linear dose-response relationship. Importantly, they highlight accelerated biological aging as a potentially modifiable contributor, an aspect largely absent from mutation specific therapies.Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, caused by a CTG trinucleotide repeat expansion in the DMPK gene that affects muscle, heart, and brain. Zafarullah et al. presented the first exploratory cerebrospinal fluid (CSF) proteomic analysis in DM1, identifying six consistently downregulated candidate biomarkers (CKAP4, SCARF1, NCAM1, CD59, PTH1R, and CA4) and a 15-protein LASSO signature implicating IGF transport, MAPK, and NCAM signaling. Together, these data establish the first CSF proteomic fingerprint for DM1.Spinal muscular atrophy (SMA) is a progressive autosomal recessive NMD caused by loss-of-function mutations in the SMN1 gene, leading to degeneration of spinal motor neurons and proximal muscle weakness. Three disease modifying therapies have been approved for SMA, all aimed at increasing SMN protein levels, including nusinersen (an SMN2 antisense oligonucleotide) and risdiplam, a small molecule SMN2 splicing modifier. As treatments improve outcomes, reliable biomarkers are essential for monitoring disease course and response. Cheng et al. assessed real world functional biomarker trajectories in patients transitioning from nusinersen to risdiplam across four institutions, demonstrating through motor function score assessments that upper limb motor function was significantly improved from pretreatment baseline at the time of switch, with a sustained upward trend following conversion. Complementing these findings, a retrospective pediatric cohort study by Huang et al. identified serum creatinine as a dynamic biomarker of therapeutic response during nusinersen treatment, outperforming both the creatinine-to-cystatin C ratio and the creatinine muscle index, and supporting its use as a practical, minimally invasive monitoring tool. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons, leading to muscle weakness, respiratory failure, and loss of voluntary movement. Its rapid progression emphasizes the critical need to identify genetic triggers and functional biomarkers that enable earlier diagnosis and proactive clinical management. On the genetic front, Shen et al. identified 20 rare ERBB4 variants in 1,627 Chinese ALS patients, finding that variant carriers experience significantly earlier disease onset. A global meta-analysis confirmed a 0.83% variant frequency, with higher prevalence in Chinese and American populations, positioning ERBB4 as a prime target for population-specific screening and targeted therapies. A parallel line of inquiry by Rong et al. introduced a noninvasive surface electromyography (sEMG) framework to objectively assess bulbar motor decline in ALS. By detecting neuromuscular irregularities during syllable repetition, the tool accurately distinguished healthy controls from patients with subclinical symptoms, supporting its use for early disease detection and measurement-based care.Pompe disease (PD), caused by deficiency of lysosomal acid alphaglucosidase (GAA), the challenges and successes of enzyme replacement therapy (ERT) in lysosomal disorders. Li et al. reported a 17-year retrospective study of 68 Chinese patients with late onset PD. Mortality was substantially lower among ERT-treated patients than untreated individuals. The predominant GAA variant (W746C) was identified in 43.3% of patients, a population specific diagnostic for East Asian cohorts. Notably, glycogenin accumulation preceded autophagy marker deposition in muscle, raising the hypothesis, that glycogenin may serve as an early pathological biomarker in PD.Myasthenia gravis (MG) is an autoimmune disorder characterized by fatigable skeletal muscle weakness, primarily driven by pathogenic autoantibodies targeting the neuromuscular junction. MG pathogenesis is increasingly recognized to involve broader immune dysregulation and inflammation beyond autoantibody production, contributing to disease severity and outcomes. Lv et al. reported that serum neurofilament light chain (sNFL) levels are elevated in patients with MG and correlate with disease severity,...

Although TDP-43 pathology is found in most sporadic and familial ALS and FTD cases, other shared pathogenic mechanisms remain largely unknown. Here we show that SIRT1 levels are decreased and acetylated p53 levels are increased in iPSC-derived neurons from sALS patients and with the FTD3-causing CHMP2B mutation. Ectopic expression of SIRT1 in these patient neurons rescues neurodegeneration and reduces acetylated p53 levels. DNA damage is elevated in both sALS and FTD3 neurons, leading to increased phosphorylation of p53 at Serine 15 and elevated levels of Ku80. Knockdown of either p53 or Ku80 rescues neurodegeneration and increases SIRT1 levels in these neurons. Moreover, ectopic expression of SIRT1 or genetic knockdown of either p53 or Ku80 suppresses retinal neurodegeneration caused by FTD3-associated mutant CHMP2B protein in an in vivo Drosophila model. These findings identify a dysregulated SIRT1–p53 feedback loop as a common pathogenic mechanism and promising therapeutic target in both sporadic and familial ALS/FTD.

A six nucleotide repeat expansion in intron-1 of the C9ORF72 gene is the most common genetic mutation affecting individuals with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Bi-directional transcription of the repeat expansion generates sense and antisense repeat RNAs that can then be translated in all reading frames to produce six distinct dipeptide repeat (DPR) proteins with unique termini. The precise site of translation initiation of these proteins within the C9ORF72 repeat expansion remains elusive. We used CRISPR-Cas9 genome editing and steric-blocking antisense oligonucleotides (ASOs) to investigate the contribution of different AUG codons in the antisense repeat RNA to the production of DPR proteins, poly(GP) and poly(PR) in C9ORF72 expansion carrier motor neurons and lymphoblast cells. We then utilized ASOs targeting C9ORF72 sense repeat RNA to examine whether sense or antisense RNA is the major source of the poly(GP) protein - a question for which conflicting evidence exists. We found that these ASOs reduced the intended sense RNA target, but also the antisense RNA, thus preventing the production of poly(PR). Our data highlights the importance of the sequences preceding the antisense CCCCGG repeat expansion for the synthesis of antisense DPR proteins and supports the use of sense C9ORF72 ASOs to prevent the accumulation of both sense- and antisense-dependent DPR proteins in C9ORF72 ALS/FTD.

Summary: While motor and cortical neurons are affected in C9orf72 ALS/FTD, it remains still largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We generated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G ₄ C ₂ repeat RNA foci, exhibit reduced C9orf72 protein levels and generate dipeptide repeat proteins. Healthy control and C9orf72 iPSC-MG equivalently express microglial specific genes and display microglial functions including inflammatory cytokine release and phagocytosis of extracellular toxic cargos such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. Select C9orf72 iPSC-MG patient lines show inability to efficiently remove phagocytosed contents, suggesting dysfunction of the endosomal-lysosomal pathways. Finally, RNA sequencing revealed overall transcriptional changes in diseased microglia yet no significant differentially expressed microglial-enriched genes. These minimal differences in cellular, molecular and functional characteristics of microglial mono-cultures suggest that a diseased microenvironment is associated with microglial activation and subsequent regulation of neuronal dysfunction.

Expanded GGGGCC (G₄C₂) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G₄C₂ repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G₄C₂ repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G₄C₂ repeats in human C9ORF72-ALS/FTD neurons.