LINC complex alterations are a hallmark of sporadic and familial ALS/FTD

Riccardo Sirtori, Michelle Gregoire, Emily Potts, Alicia Collins, Liviana Donatelli, Claudia Fallini

Posted on: 3 June 2024 , updated on: 4 June 2024

Preprint posted on 13 March 2024

The missing link between ALS and nuclear deformation? It's the missing LINC

Selected by Megane Rayer, Rivka Shapiro, preLights peer support

Categories: cell biology, pathology

Written by: Rivka Shapiro

Physics and Molecular Biology Student at Yeshiva University

Background

What do we know about ALS?

Amyotrophic lateral sclerosis, commonly referred to as ALS, is a neurodegenerative disease affecting motor neurons. It results in the loss of motor control in affected patients, diminishing their capacity for movement and ultimately causing death. There are two types of ALS: familial, which appears to have a heritable genetic cause, and sporadic, which does not. A significant portion of familial cases, as well as a few sporadic cases, are linked to mutations in the C9ORF72 gene (C9-ALS). [4]

What is the LINC Complex?

The Linker of Nucleoskeleton and Cytoskeleton (LINC} complex is a multiprotein structure spanning the nuclear envelope, connecting the nucleoskeleton and cytoskeleton. It is mainly composed of trimers of two categories of proteins, SUNs and Nesprins. The LINC complex transmits force to the nucleus, thereby affecting its size, shape, position within the cell and chromatin organization. [2].

What is the connection between these two topics?

It has been previously shown that ALS-affected tissue exhibits abnormal nuclear morphology [1][3]. The authors of this preprint recently demonstrated that pathogenic expression of the C9ORF72 gene is affected by nuclear envelope homeostasis [4]. With this in mind, the LINC complex is a logical candidate to study as it transverses the nuclear envelope and mediates mechanical stress.

Key Findings

LINC components are reduced and mislocalized in C9-ALS neurons

The authors studied three model neurons bearing a pathogenic mutation of C9, known to be associated with ALS. First, they used a 2D tissue culture model of iPSC-derived motor and cortical neurons. They identified a reduction in SUN1, SUN2, and nesprins in the affected cells compared to the controls using immunofluorescent (IF) staining as well as blotting the cell lysates.

In control cells, these proteins were localized to the nuclear envelope in a pattern suggestive of LINC complexes. However, this was not the case in cells bearing the C9 mutation. In addition, both SUN and Nesprin were less abundant in the nucleus compared to the cytoplasm in the affected cell groups.

The authors then investigated in 3D tissue culture, using iPSC-derived spinal cord organoids. Organoids with the C9 mutation showed reduced and mislocalized SUN.

Finally, postmortem biopsies from patients with both a confirmed ALS diagnosis and C9 mutation were also studied using IF. These patient samples confirmed the results of the previous models: SUN is mislocalized from the nuclear envelope, and reduced in abundance compared to controls.

Abnormal nuclear morphology is correlated with SUN mislocalization

Because LINC affects nuclear morphology, the authors investigated these variables in the three models detailed above. They found that in the organoids, aberrant SUN presence was the driving factor for any nuclear and nucleolar abnormalities. In the biopsy tissue, C9-ALS neurons with normal SUN distribution did not have nuclear defects, but those with SUN mislocalization did.

My Opinion

Furthering our understanding of possible mechanisms driving the pathogenesis of ALS (and other neurodegenerative disease) is crucial in the search for treatments. The notable presence of nuclear morphological changes, and the newly-suspected role of the nuclear envelope in ALS indicate that mechanical forces acting on the nucleus may play a role in early ALS pathogenesis.

The preprint authors take a holistic approach in their use of three different cell models. They acknowledge the strengths and limitations of each method, and are able to combine their results into a compelling case for proving the role of LINC complexes in ALS. The preprint is particularly interesting to the lab where I am currently doing my internship, as we investigate the role of LINC in Alzheimer’s, another neurodegenerative disease.

Questions

Q1. Do you have an idea why 2D-cultured cells did not show significant change in nuclear morphology after LINC disruption?

Q2. What could be the cause for the difference in mislocalization and reduced abundance of SUN1 compared to SUN2?

Q3. Not all cases of ALS are caused by the C9 mutation – have you investigated nuclear morphology or LINC component abundance in samples without the mutation? If not, what do you think you may find if you do?

References

Aladesuyi Arogundade, O. et al. Nucleolar stress in C9orf72 and sporadic ALS spinal motor neurons precedes TDP-43 mislocalization. Acta Neuropathol. Commun. 9, 1–16 (2021).
Alam, S. G. et al. The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity. Sci. Rep. (2016) doi:10.1038/srep38063.
Baskerville, Victoria, Sampath Rapuri, Emma Mehlhop, and Alyssa N. Coyne. “SUN1 facilitates CHMP7 nuclear influx and injury cascades in sporadic amyotrophic lateral sclerosis.” Brain 147, no. 1 (2024): 109-121.
Riccardo Sirtori, Michelle Gregoire, Alicia Collins, Serena Santangelo, B. & Chatragadda, Robert Cullen, Antonia Ratti, C. F. Altered nuclear envelope homeostasis is a key pathogenic event in C9ORF72-linked ALS/FTD. 1, (2024)

Tags: als, linc complex, motor neurons, nuclear shape, nucleus

doi: https://doi.org/10.1242/prelights.37561

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Author's response

Claudia Fallini shared

A1. This is a great question! The regulation of nuclear and nucleolar size and morphology is a complex process, and many intrinsic and extrinsic factors contribute to it, including the LINC complex and actin cytoskeleton. We think that the mechanical stress on cells cultured on glass, a really hard substrate with an elastic modulus of about 70 Giga Pascals (GPa) is so high that it may overshadow any effect from other cellular players. For comparison, the hardest biological substrate is bone, with a stiffness that ranges between 10 MPa and 3 GPa. In organoids, cells grow in a more physiological environment with reduced mechanical stress compared to 2D monocultures, making them more sensitive to the effects of intracellular signals and modulators.

A2. This is another important question that we are looking to find an answer to. SUN1 and SUN2, the inner nuclear membrane component of the LINC complex, are a part of a much larger and complex mechano-transduction system that involves a large number of nuclear envelope proteins. In addition, both SUN proteins have been proposed to have additional functions outside of the LINC complex. The differences in their levels and localization may thus be connected to other nuclear processes, including regulation of nucleocytoplasmic transport and chromatin organization.

A3. Some of the postmortem tissue samples we used in the paper are from sporadic ALS patients. While the sample size is not large, the data show no major difference in the LINC or nuclear phenotype between the two groups. We are currently looking at expanding our sample collection including other non-C9 familial ALS cases. One gene we are particularly interested in is SOD1, since nuclear pore defects and TDP-43 aggregates don’t seem to be typical pathological sings in patients with these mutations.

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