23 Feb 2024

The pathological accumulation of TDP-43 that underlies many cases of FTD, ALS, and LATE-NC has been notoriously difficult to recapitulate in mice or cultured human cells. In a paper published February 14 in Nature, scientists led by Magdalini Polymenidou of the University of Zurich describe a culture method that they believe more closely models what happens in disease. Starting with induced pluripotent stem cells (iPSCs), the researchers selected and cloned neural stem cells. This added selection step results in homogenous, self-renewing cultures, which yielded a variety of glia and neuron cell types that forged functional and synaptic connections. These “iNet” cultures are longer-lived and their cells more mature than those developed from other iPSC-based techniques.

Using iNets to model TDP-43 pathology, the authors found that overexpressing the RNA binding protein in a minuscule fraction of neurons provoked TDP-43 inclusions in their neuronal neighbors. Gene regulation changed dramatically, with a notable uptick in expression of the synaptic signaling protein NPTX2. Neurotoxicity ensued, casting the synaptic protein as an executioner downstream of TDP-43 pathology.

TDP-43 micromanages RNA within the nucleus, where it latches onto numerous transcripts, steering their splicing and expression. In FTD/ALS, TDP-43 gets ensnared in cytoplasmic inclusions, shirking its nuclear duties and leading to widespread transcriptomic snafus. Some affected transcripts, such as stathmin-2, are dysregulated only in human cells (Jan 2019 news), so researchers deploy human iPSC-derived neuronal cultures to study TDP-43 pathology. Alas, these stem cell cultures typically lack the classic TDP-43 inclusions found in postmortem brain samples from people with FTD/ALS. Perhaps cells in these cultures die before they can face the consequences of TDP-43 dysregulation.

To get around this problem, first author Marian Hruska-Plochan and colleagues used iPSC colony morphology as a guide to manually select radially arranged groups of neural stem cells (NSCs), called rosettes, for expansion (Bohaciakova et al., 2019). In so doing, they eliminated contaminating cells, and made the NSC cultures purer and healthier. The resulting clonal lines—called induced colony morphology-NSCs (iCoMo-NSCs)—could then be efficiently and consistently differentiated into all manner of brain cell types.

By adding the just-right mix of growth factors, the scientists differentiated iCoMo-NSCs into iNets. As their name implies, these form functional networks of neural cells that buzz with electrophysiologically active circuits. Cell types ranging from excitatory to inhibitory neurons, astrocytes, and oligodendrocytes thrived in the cultures, where they blossomed into functionally mature cells over several months. Microglia are not expected because they come from a different cell lineage.

Single-cell RNA sequencing indicated that the neuronal and glial inhabitants of iNets boasted a similar repertoire of mature brain cell types as cortical brain organoids, which use a three-dimensional matrix to coax organization of neural cells into a somewhat brain-like structure (Aug 2013 news; Jun 2019 news). Relative to organoids, iNets are easier to manipulate, clone, and image, Polymenidou emphasized, noting that her method combines the brain-like features of organoids with the experimental pliability of iPSC-derived monocultures.

What Role TDP-43?
The researchers used these iNets to investigate the effects of TDP-43 aggregation. Only about 2 percent of cortical neurons show signs of this pathology in postmortem samples from people with FTLD (Liu et al., 2020). To simulate this, the researchers transduced young iNet cultures using a low titer of lentiviral vectors carrying a tagged version of wild-type TDP-43 under control of an inducible promoter. Switching it on boosted TDP-43 expression two- to threefold. Within the first three weeks of expression, half the transduced cells died.

Analysis of iNet lysates indicated that the tagged TDP-43 had become increasingly insoluble over time, aggregating into high molecular weight forms akin to those found in TDP-43 proteinopathies. Despite this, nary an inclusion could be seen in the transduced cells. Instead, small, dot-like inclusions arose in the soma of neighboring, non-transduced neurons. These specks grew into larger deposits that extended into neuronal processes (image below). Why inclusions did not develop in the cells overexpressing the protein, and how their neighboring cells managed to acquire them, remain to be seen. Polymenidou thinks that when neurons burdened by TDP-43 overexpression start dying off, neighboring neurons might take up debris, including TDP-43 aggregates. Alternatively, signals from the stressed neurons somehow incite TDP-43 pathology within other cells, she speculated. Similar questions are asked about the spread of other proteopathic proteins such as Aβ and tau (Walsh and Selkoe, 2016; Nov 2021 news).

Given TDP-43’s role in splicing and regulating RNA transcripts, the researchers used single-cell RNA sequencing to investigate how its overexpression and aggregation might influence the transcriptomes of iNet cells. In iNets bestowed with extra TDP-43, the scientists identified 17 transcriptional clusters of cells, including 12 neuronal ones. One of these was made up almost entirely of neurons overexpressing TDP-43, which altered the abundance of many transcripts, particularly those known to interact with TDP-43. Notably, these cells possessed scant transcripts encoding the microtubule-binding protein stathmin-2 and the axonal protein UNC13A, known clients of TDP-43’s exon removal service. Previous studies have demonstrated that without functional TDP-43, these transcripts retain cryptic exons, which reduces their expression and leads to their drop in the CNS of people with FTD/ALS (Melamed et al., 2019; Klim et al., 2019; Apr 2021 news).

Traveling TDP-43? After five weeks of TDP-43 overexpression in iNets (left panels), dot-like inclusions of phosphorylated TDP-43 (yellow) were spotted in neurons that did not overexpress tagged TDP-43 (green). By nine weeks, larger aggregates had extended beyond the soma into neuronal processes (right panels). [Courtesy of Hruska-Plochan et al., Nature, 2024.]

Among the most upregulated transcripts in TDP-43-overexpressing neurons was neuronal pentraxin-2 (NPTX2), a synaptic protein that can be secreted. Curiously, NPTX2 protein reportedly plummets in the CSF of people with neurodegenerative diseases including AD and FTD, suggesting that it reflects synaptic deterioration (Xiao et al., 2017; Aug 2019 conference news; van der Ende et al., 2020; Watson et al., 2023).

How might a glut of TDP-43 lead to a boost in NPTX2 transcripts? The scientists found that under physiological conditions, TDP-43 binds the 3' UTR, which they believe might compromise the stability or transport of the transcript. With TDP-43 bound up in insoluble clumps, NPTX2 translation goes unchecked. This protein enhances glutamate receptors, and glutamate excitotoxicity is a suspect as a pathological pathway in ALS and other neurodegenerative diseases, suggesting a gain-of-function toxicity.

Indeed, knocking down NPTX2 in iNets slightly lessened the toxicity associated with neuronal TDP-43 overexpression in the case of one of two shRNA constructs. The researchers also found inclusions of NPTX2 in cells that overexpressed TDP-43 in their iNets. The same was true in postmortem brain samples from people with TDP-43 proteinopathy, including FTLD, ALS, or AD—inclusions of NPTX2 and TDP-43 occurred in the same cell, though separately. Together, the findings suggested that TDP-43 dysfunction unleashes NPTX2 expression, leading to its aggregation and potentially derailing its function. To Nicolas Seyfried of Emory University in Atlanta, Polymenidou’s findings hint at an alternative mechanism for loss of NPTX2 in the CSF of people with AD and FTD. “This study opens up possibility that NPTX2 is not getting into the CSF because it’s aggregated inside of the cells,” Seyfried said.

More studies are needed to determine if TDP-43 pathology causes CSF NPTX2 to wane in people with AD.

Emanuele Buratti of the International Centre for Genetic Engineering and Biotechnology in Trieste, Italy, co-author on one of the studies linking TDP-43 dysfunction with the retention of cryptic exons in UNC13A, commented that the new study adds NPTX2 to a short list of proteins that may be responsible for the toxicity following TDP-43 loss of function in the nucleus. “This exciting result suggests that many misregulated events can probably be compensated by cellular mechanisms, and therefore, from a therapeutic point of view, it might be advantageous to just focus on a set of key factors such as UNC13A, STMN2 and now, of course, NPTX2, and just let the rest take care of themselves.” 

Polymenidou told Alzforum that her lab is in the process of generating iCoMo-NSC lines from people with other neurodegenerative diseases, as well as lines in which disease-related mutations have been introduced with CRISPR. These lines will be shared with other researchers, she said.—Jessica Shugart

Comments

1. • Emanuele Buratti
     International Centre for Genetic Engineering and Biotechnology
   • Posted: 23 Feb 2024

   One great drawback when studying proteins like TDP-43 is the incredible number of genes whose expression is controlled by this protein, and which become misregulated following TDP-43 aggregation in pathological conditions. From a therapeutic point of view, this observation raises the question as to whether it will be necessary to rescue all or most of these misregulated genes in order to prevent neuronal death, or whether it might be sufficient to focus on just a handful of key genes.

   Using state-of-the-art approaches, the Polymenidou lab has shown that a single factor, called NPTX2, may be responsible for much of the toxicity displayed by TDP-43 loss of function in the nucleus. This exciting result suggests that many misregulated events can probably be compensated for by cellular mechanisms. Therefore, from a therapeutic point of view, it might be advantageous to focus on a set of key factors such as UNC13A, STMN2 and now, of course, NPTX2, and let the rest take care of themselves. Or, to paraphrase a quote from Gorge Orwell’s Animal Farm: “In neurodegeneration, all consequences of TDP-43 aggregation are equal, but some are more equal than others.”

   View all comments by Emanuele Buratti

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References

News Citations

1. Microtubule Regulator Connects TDP-43 to Axonal Dysfunction 18 Jan 2019
2. Mini Brain in a Dish Models Human Development 30 Aug 2013
3. Reproducible Brain Organoids Could Offer New Models for Research 6 Jun 2019
4. Doubling of Tau Seeds, Not Spread, Sets Pace of Tauopathy in Alzheimer's 3 Nov 2021
5. Sans Nuclear TDP-43, Splicing of An ALS/FTD Gene Goes Awry 25 Apr 2021
6. Synaptic Proteins in CSF: New Markers of Cognitive Decline? 22 Aug 2019

Paper Citations

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5. Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. 2019 Feb;22(2):167-179. Epub 2019 Jan 14 PubMed.
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7. van der Ende EL, Xiao M, Xu D, Poos JM, Panman JL, Jiskoot LC, Meeter LH, Dopper EG, Papma JM, Heller C, Convery R, Moore K, Bocchetta M, Neason M, Peakman G, Cash DM, Teunissen CE, Graff C, Synofzik M, Moreno F, Finger E, Sánchez-Valle R, Vandenberghe R, Laforce R Jr, Masellis M, Tartaglia MC, Rowe JB, Butler CR, Ducharme S, Gerhard A, Danek A, Levin J, Pijnenburg YA, Otto M, Borroni B, Tagliavini F, de Mendonca A, Santana I, Galimberti D, Seelaar H, Rohrer JD, Worley PF, van Swieten JC, Genetic Frontotemporal Dementia Initiative (GENFI). Neuronal pentraxin 2: a synapse-derived CSF biomarker in genetic frontotemporal dementia. J Neurol Neurosurg Psychiatry. 2020 Jun;91(6):612-621. Epub 2020 Apr 9 PubMed.
8. Watson CM, Dammer EB, Ping L, Duong DM, Modeste E, Carter EK, Johnson EC, Levey AI, Lah JJ, Roberts BR, Seyfried NT. Quantitative Mass Spectrometry Analysis of Cerebrospinal Fluid Protein Biomarkers in Alzheimer's Disease. Sci Data. 2023 May 9;10(1):261. PubMed.

Further Reading

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