Background and Aims:

Motor neuron diseases (MNDs), including amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal and bulbar muscular atrophy (SBMA), are a heterogeneous group of neurodegenerative disorders characterized by motor neuron loss. Despite variability in onset, progression and genetic causes, these disorders share a common involvement of skeletal muscle that is suspected to have a relevant role in MND pathogenesis.

Due to the key role of muscle-specific microRNAs (myomiRs) in muscle development, here we investigated the expression of miR-206, miR-133a, miR-133b and miR-1 myomiRs, and their target genes, in G93A-SOD1 ALS, Δ7SMA and KI-SBMA mouse muscle during disease progression.

Methods:

MyomiRs and their putative target genes, PAX7, MYOD, MYOG, and MEF2, were analyzed by qPCR in ALS-G93A-SOD1, Δ7SMA-SMA and AR113Q-SBMA mice during disease progression. Sperman’s correlation analyses were performed to examine the myomiR/mRNA relationships. The corresponding myomiR target proteins were analysed by western blot. By qPCR, myomiRs were assessed in serum of 14 ALS-SOD1, 23 SMA pediatric, 10 SBMA patients, and 30 controls, including 19 pediatric patients with encephalitis.

Results:

We revealed myomiRs/mRNA target gene dysregulation as common pathogenic feature of the three MNDs. A similar myomiR signature was observed in serum from SOD1-mutated ALS, SMA and SBMA patients, with miR-206 up-regulation being identified in both animal muscle tissues and patients’sera.

Conclusions:

Our findings highlight the role of myomiRs as contributing factors and promising non-invasive biomarkers in ALS, SMA and SBMA, laying the groundwork for further investigations to explore myomiR potential as future therapeutic targets for MNDs.

Background and Aims:

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. Underlying genetic pathomechanisms include the C9orf72 repeat expansion, the most frequent genetic cause of ALS (C9ALS) in Western countries. Despite recent progress in unraveling C9ALS pathogenesis, reliable disease models and disease-modifying therapies still lack. Here, we aim to model C9ALS in vitro using 3D human spinal cord organoids (SCOs).

Methods:

We differentiated C9ALS induced pluripotent stem cells (iPSCs) and isogenic controls using a free-floating 3D-culture method. We generated SCOs with a modified Lancaster’s protocol promoting neural caudalization and ventralization. Then, we treated C9ALS SCOs with morpholino antisense oligonucleotides (MO) against C9Orf72 repeat expansion. Finally, we assessed the differentiation of organoids at different time points with morphological, immunohistochemical, and qPCR analysis.

Results:

We generated isogenic and C9ALS SCOs exhibiting different co-existing neuronal subpopulations. SCOs expressed neural progenitor, pan-neuronal, astrocyte, motor neuron, and rostrocaudal markers, including markers of cervicobrachial spinal cells. Compared to controls, C9ALS early SCOs diameters measured from day 2 to day 13 were significantly reduced. C9ALS organoids displayed increased dipeptide repeat proteins (DPRs) levels, DNA damage markers associated with C9orf72 expansion, and cytoplasmic inclusions of translocated TDP-43, C9ALS-specific disease hallmarks. Gene expression analysis using qPCR reported differential expression of genes associated with DNA damage and motor neurons in MO-treated C9ALS organoids.

Conclusions:

SCOs represent a valuable system for modeling features of C9ALS pathology, investigating C9ALS pathomechanisms, and testing possible new treatments in vitro.