Embryonic Motor Neuron Programming Factors Reactivate Immature Gene Expression and Suppress ALS Pathologies in Postnatal Motor Neurons

Neurology Bioengineering Basic Medical Sciences Cell Biology Biochemistry Genetics Life Sciences Medicine
motor neuron ALS transcription factors gene expression neurodegeneration reprogramming gene therapy

1. Academic Background and Research Motivation

Degenerative diseases of motor neurons, such as Amyotrophic Lateral Sclerosis (ALS), have long been a key research area in neuroscience. ALS is characterized by adult onset, with progressive degeneration of motor neurons leading to paralysis and death. In diseases such as ALS, aging is considered a major risk factor, but the molecular mechanisms underlying why mature motor neurons are susceptible to pathological damage, while young motor neurons can resist such insults, remain unclear. Previous research has shown that as motor neurons mature, their gene expression and chromatin structure change dramatically, with about 7,000 genes and 100,000 chromatin accessibility regions showing substantial alterations during maturation.

The research team noted that embryonic motor neurons possess strong resistance and regenerative potential, which is gradually lost later in life. They proposed an important hypothesis: If mature motor neurons could be made to re-express embryonic “selector transcription factors,” such as Isl1 and Lhx3, could the neurons’ vitality be restored and could ALS pathology be delayed or blocked? Isl1 and Lhx3 play core roles in the generation and differentiation of embryonic motor neurons but are downregulated after birth.

This study aims to address whether re-expressing these embryonic transcription factors in mature neurons can restore a youthful gene expression state and thus alleviate or prevent pathological damage in ALS mouse models. This strategy not only involves foundational science but also offers a novel therapeutic idea for ALS and other neurodegenerative diseases.

2. Article Source and Author Information

The paper, titled “Embryonic motor neuron programming factors reactivate immature gene expression and suppress ALS pathologies in postnatal motor neurons,” was published in Nature Neuroscience, Volume 28, October 2025 (pp. 2044–2053). The main authors include Emily R. Lowry, Tulsi Patel, Jonathon A. Costa, Elizabeth Chang, and others. The team is primarily affiliated with Columbia University Irving Medical Center (USA), and some authors are now at Rutgers University Robert Wood Johnson Medical School. Multiple team members contributed equally, exemplifying a multidisciplinary collaboration at Columbia University.

3. Research Workflow and Technical Innovations

1. Overview of the Workflow

The research includes the following main sections:

  • Construction and validation of a motor neuron-specific viral tool;
  • Re-expression of Isl1 and Lhx3 in the ALS mouse model;
  • Single-nucleus multiomic sequencing analysis (multiome RNA and ATAC-seq) to examine gene expression and chromatin structure changes;
  • Assessment of key pathological markers (such as SQSTM1 round bodies, SOD1 pathological structures) and behavior symptoms;
  • Validation of long-term neuroprotective effects in motor neurons;
  • Functional validation at the molecular and tissue levels.

2. Construction of a Motor Neuron-Specific AAV Expression System

The team utilized the knowledge that motor neurons express choline acetyltransferase (CHAT) and chose the region 3kb upstream of the CHAT gene, the “chAT enhancer” (abbreviated as chate), to screen and validate its chromatin accessibility and transcriptional specificity during motor neuron maturation. In preliminary experiments, the researchers cloned the chate sequence into an AAV vector and drove mCherry expression, with intracerebroventricular injections into neonatal rodents. They observed highly efficient and specific mCherry expression in motor neurons. Compared to a ubiquitous promoter (e.g., CAGGS-driven GFP), chate-driven AAV only expressed in motor neurons, without spillover to other cholinergic cells.

3. Re-expression of Isl1 and Lhx3 in the ALS Mouse Model

The study used the classic mouse model for amyotrophic lateral sclerosis: SOD1^G93A transgenic mice. Neonatal mice (P1) received intracerebroventricular injections of AAVs carrying human Isl1 and Lhx3; the efficiency and stability of transcription factor re-expression were tracked under different viral titers. Under high-titer conditions, the vast majority of motor neurons successfully re-expressed Isl1 or Lhx3, maintaining expression for several weeks.

4. Single-Nucleus Multiomic Analysis: Gene Expression and Chromatin Structure

The team used CHAT-Cre/SUN1-GFP reporter mice, purified nuclei using cell sorting, and performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC-seq with the 10x multiome platform, capturing both transcriptomic and chromatin accessibility data. To distinguish virally expressed Isl1/Lhx3, they created a custom reference genome containing viral elements (human Isl1, Lhx3, chimeric intron, WPRE). Motor neuron samples collected at P21 post-treatment were clustered and expression characteristics were analyzed with standard analysis workflows.

5. Data Processing and Algorithmic Innovations

They primarily used the Seurat and Signac packages for data processing, applying Canonical Correlation Analysis (CCA) to integrate and cluster multiomic datasets. The OMICS data allowed them to identify motor neuron subtypes (Alpha, Gamma, Type 3) and to dissect the dynamic changes in virally driven gene expression among these subtypes.

6. Histopathological and Behavioral Assessments

In the ALS mouse model, spinal cord tissue samples were collected at P45 (early stage), P75 (progressive stage), and P120 (late stage). Immunostaining was used to detect key pathological markers such as “SQSTM1 round bodies” and abnormal SOD1 structures. Simultaneously, behavioral tracking assessed clinical symptoms such as fine hindlimb tremors and overall survival.

4. Detailed Main Results

1. Isl1 and Lhx3 Re-expression Partially Reverts Motor Neurons to an Immature State

In the AAV–Isl1+AAV–Lhx3 group, cohort analysis showed that in about half of the motor neurons, the specific marker MNX1 (highly expressed in embryonic motor neurons) was reactivated. In contrast, MNX1 expression was nearly absent in the control group, indicating Isl1 and Lhx3 re-expression can indeed reboot the embryonic gene expression program.

Further single-nucleus sequencing revealed that in the Alpha and Type 3 motor neuron subtypes, the virus-expressing groups formed new clusters (“Alpha prime,” “Type 3 prime”) distinct from the controls, with noticeable changes in gene expression and chromatin accessibility. Chromatin accessibility and regulatory elements were enriched at Lhx3 binding sites (Homeodomain motif), suggesting that these two factors not only directly activate target genes but also alter chromatin state, promoting a global ‘rejuvenation’ of gene expression.

2. Highly Subtype-Selective Effects Among Motor Neuron Subtypes

Although Isl1 and Lhx3 could be expressed in all three major motor neuron subtypes, actual gene expression changes were significant only in the Alpha and Type 3 subtypes. Gamma motor neurons showed minimal changes, indicating sharply different subtype responsiveness to selector transcription factors, possibly due to intrinsic chromatin accessibility or differences in endogenous regulatory factors.

3. Rejuvenated Gene Expression Improves Neuronal Response to ALS Pathology

In the SOD1^G93A mouse model, one of the earliest pathological markers is the formation of SQSTM1 round bodies, associated with dysfunction in the protein degradation system. Upon re-expression of Isl1 and Lhx3, round body formation significantly decreased (in high-titer conditions, over 80% of transgene-expressing motor neurons could clear this pathology) without affecting overall motor neuron number.

As disease progressed, SOD1 pathological structures accumulated, but Isl1 and Lhx3 expression could markedly reduce their frequency. Quantitative assays showed that pathological SOD1+ structures at P75 were reduced to a third of the control group, and transcriptomic/protein data indicate that this effect is due to mitigation of damage, not gene knockdown.

4. Delayed Behavioral Symptoms and Improved Motor Neuron Survival

Under low-titer experimental conditions where only 20% of motor neurons expressed the transgene, behavioral analysis revealed a significant delay in the onset of fine hindlimb tremors (from P90 to P105 in females), though survival was not extended. In the high-titer group, the number of surviving motor neurons at P120 increased significantly, with the proportion of cells co-expressing Isl1 and Lhx3 unexpectedly maintained or increased, suggesting that this rejuvenation treatment provides long-term neuronal protection.

Additionally, the team confirmed that under high-titer AAV, the relationship between Isl1–Lhx3 expression and pathological protection is tied to the stability of transcription factor expression and cell selectivity.

5. Research Conclusions and Scientific Value

Through comprehensive experimental evidence, the team demonstrates for the first time that heterochronic re-expression of embryonic selector transcription factors in mature motor neurons can induce cell subtype-specific changes in gene expression by ‘transcriptional rejuvenation,’ successfully reducing early- and late-stage pathological damage in ALS mouse models, delaying onset of behavioral symptoms, and improving motor neuron survival. This strategy differs from traditional cell reprogramming methods (such as general pluripotency factor-driven approaches) by implementing targeted rejuvenation in susceptible cell types, achieving a precision therapeutic approach.

Scientifically, the study reveals the relationship between genomics changes during motor neuron maturation and their pathological susceptibility, providing a theoretical foundation for exploring cell type-selective interventions. In terms of application, the research opens a potential pathway for intervention in adult-onset neurodegenerative diseases such as ALS—delivering targeted transcription factors to restore cell resistance to stress, and holding promise for the development of innovative gene therapy approaches.

6. Research Highlights and Innovations

  1. Cell Type–Precise Reprogramming: Unlike traditional generalized reprogramming, this study used a motor neuron-specific enhancer to precisely activate selector transcription factors, improving cellular specificity and safety.
  2. Application of Multiomic Technologies: Integrating single-nucleus RNA and ATAC-seq multiomic platforms, the work comprehensively reveals coordinated changes in gene expression and chromatin accessibility, providing multidimensional evidence for understanding cell fate.
  3. Subtype-Selective Effects and Mechanisms: The study discovers and analyzes the differential responses of motor neuron subtypes to transcription factors, highlighting the molecular basis of cell-intrinsic susceptibility and providing key insight into selective vulnerability in ALS.
  4. Validation in Disease Models: With continuous multi-stage tracking in the ALS animal model, and integration of molecular, cell, tissue, and behavioral data, the research lays a solid foundation for translational application.

7. Other Important Information

This study also addresses practical issues, such as the stability of transcription factor expression and the impact of viral titer adjustments on disease protection. The team suggests that future work should aim to improve the durability of expression and to explore whether late-stage intervention remains effective. Some open questions are raised, such as potential mechanisms by which modulation of motor neuron excitability improves protein homeostasis, and the use of single versus combined transcription factors.

8. Conclusion and Outlook

This research, through an innovative cellular rejuvenation strategy, precisely activates endogenous protective programs in motor neurons, effectively mitigating ALS-related pathology and bringing a highly promising new direction for the treatment of neurodegenerative diseases. In the future, the applicability and safety of this strategy in other related diseases can be further explored, advancing the development of precision and personalized medicine.