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Amyotrophic lateral sclerosis associated mislocalisation of TDP-43 to the cytoplasm causes cortical hyperexcitability and reduced excitatory neurotransmission in the motor cortex

MS Dyer, KE Lewis, AK Walker, TC Dickson, A Woodhouse, CA Blizzard

Preprint posted on 12 June 2020 https://www.biorxiv.org/content/10.1101/2020.06.11.147439v1

Article now published in Journal of Neurochemistry at http://dx.doi.org/10.1111/jnc.15214

Linking neuronal hyperexcitability and TDP-43 pathology: a step towards combining the ALS puzzle pieces

Selected by Kristina Kuhbandner

Categories: cell biology, neuroscience

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, especially affecting motor neurons in the cortex and spinal cord, thus resulting in progressive paralysis and ultimately respiratory failure. Despite substantial research efforts, it is still elusive why these neurons are particularly vulnerable, and effective treatment is lacking. Although being a very heterogenous disease, TDP-43 (TAR-DNA binding protein of 43 kDa) aggregations are observed in more than 90% of patients and thus considered as major pathological hallmark (1). This ubiquitous and mostly nuclear protein has an essential role in RNA metabolism and under physiological conditions, TDP-43 levels and localization are tightly regulated. Pathological TDP-43 in contrast often is abnormally cleaved, phosphorylated or ubiquitinylated and accumulates in the cytoplasm (2).

Another well described feature is neuronal hyperexcitability which has been observed in ALS patients and also can be recapitulated in mouse models of familial ALS (3-5). Of note, these disturbances in motor cortex excitability occur prior to symptom onset and may be a predictor of disease progression (5, 6).

Strikingly, up to date, not much is known about the relationship of these two prevalent disease characteristics. Therefore, the present study aimed to investigate the impact of TDP-43 mislocalization on neuronal activity.

Experimental approach

To model TDP-43 mislocalization in the forebrain, transgenic mice with inducible overexpression of human TDP-43 lacking a functional nuclear localization sequence (hTDP-43-ΔNLS) were used. The expression of the transgene was driven by a neuron-specific promotor and temporally regulated by the tetracycline transactivator system (7). Using the same model, wildtype (WT) hTDP-43 with a functional NLS was expressed to determine whether also increased levels of hTDP-43 can impact excitability. Expression of hTDP-43 was initiated by removal of doxycycline from the diet 30 days after birth and experiments were performed about 30 days after transgene induction.

Key findings of the study

First, the authors showed that mislocalization or overexpression of hTDP-43 did not result in increased neuronal cell death in layer V or IV. However, motor neurons expressing hTDP-43-ΔNLS exhibited altered electrophysiological membrane properties. These were accompanied by hyperexcitability evident in increased firing rates, lower rheobase and decreased difference between the resting membrane potential and the action potential (AP) threshold. Interestingly, overexpression of WT hTDP-43 did not alter any of the assessed electrophysiological parameters. Similarly, subtle changes in AP characteristics, including afterhyperpolarization amplitude and AP rise time were present in motor neurons with cytoplasmic localization of hTDP-43, but not in cells overexpressing WT hTDP-43. Given that aberrant synaptic function is reported as ALS feature, finally the effect of mislocalization on excitatory inputs was analyzed. The frequency of mini excitatory post-synaptic currents to hTDP-43-ΔNLS layer V pyramidal neurons as well as the frequency of excitatory post-synaptic currents was lower compared to control cells. This indicates that cytoplasmic hTDP-43 is linked to a loss of excitatory neurotransmission to motor neurons.

Overall this preprint provides evidence that mislocalization of hTDP-43 rather than overexpression drives hyperexcitability and synaptic dysfunction in pyramidal neurons of the motor cortex, thereby contributing to the profound understanding of the relationship between these common ALS features.

Why I chose this paper

ALS is a very heterogenous and multifactorial disease, thus tremendously complicating the identification of effective treatment strategies. In the past years, most studies aimed at analyzing isolated aspects of disease pathology, thereby adding knowledge to basic disease mechanisms. However, in my opinion, it is time also trying to combine the individual pieces of the puzzle by investigating the interplay of different common pathological characteristics. I particularly like the present study as it meets this need by investigating the interaction of TDP-43 pathology and neuronal activity, simultaneously underlining the complexity of the disease.

Future directions

ALS is a rapidly progressing disorder and patients may only have a few months from first onset of symptoms to the characteristic development of detrimental paralyzing features. Thus, it is critical to define the pathological events occurring before and at very early stages of the disease. Correcting disturbances in neuronal excitability, which is also one of the proposed actions of riluzole, an approved ALS drug, seems to be a promising therapy option. However, at later disease stages, excitability might be reduced which may also explain diminished riluzole effectiveness with increased disease duration (8). Therefore, analyzing the impact of TDP-43 mislocalization on neuronal excitability at different time points/disease stages is of major interest. To address this issue, time points of analysis after inducing the expression of hTDP-43-ΔNLS could be varied.

Strikingly, very recently Weskamp et al., reported that increased neuronal activity leads to an upregulation of truncated TDP-43 isoforms, which aggregated in cytoplasmic inclusions also sequestering full-length TDP-43 (9). These findings provide further evidence for a link between hyperexcitability and TDP-43 pathology and suggest the existence of a vicious circle, in which hyperexcitability and cytoplasmic TDP-43 accumulation can potentiate each other. It remains to be determined whether TDP-43 mislocalization is a cause or consequence of hyperexcitability or if both emerge independently.

In conclusion, unraveling the link between hyperexcitability and TDP-43 mislocalization is an important step towards understanding the various processes involved in disease pathogenesis, but also further increases the complexity of the puzzle.

Questions to the authors

1) Extranuclear TDP-43 is known to be involved in stress-granule formation and shows the propensity to form aggregates. Did the authors observe respective TDP-43 assemblies in layer V cortical neurons with TDP-43 mislocalization showing abnormal excitability?

2) Using the same mouse model, it has previously been reported that TDP-43 mislocalization induced by the lack of the NLS results in a significant loss of neurons (9). However, in the motor cortex assessed in this study, no overt cell death was recognized. Do you have any explanation for this discrepancy?

3) Did you observe any phenotypical changes in animals showing hyperexcitability?

4) It is still under debate whether and to what extend depletion of TDP-43 from the nucleus or presence of TDP-43 in the cytoplasm contribute to TDP-43 toxicity. Does mislocalization of hTDP-43 affect TDP-43 levels in the nucleus in your model?

References

  1. Mackenzie, Ian RA, et al. “Pathological TDP‐43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations.” Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society 61.5 (2007): 427-434.
  2. Chen-Plotkin, Alice S., Virginia M-Y. Lee, and John Q. Trojanowski. “TAR DNA-binding protein 43 in neurodegenerative disease.” Nature Reviews Neurology 6.4 (2010): 211.
  3. Fogarty, Matthew J., Peter G. Noakes, and Mark C. Bellingham. “Motor cortex layer V pyramidal neurons exhibit dendritic regression, spine loss, and increased synaptic excitation in the presymptomatic hSOD1G93A mouse model of amyotrophic lateral sclerosis.” Journal of Neuroscience 35.2 (2015): 643-647.
  4. Saba, L., et al. “Altered functionality, morphology, and vesicular glutamate transporter expression of cortical motor neurons from a presymptomatic mouse model of amyotrophic lateral sclerosis.” Cerebral cortex 26.4 (2016): 1512-1528.
  5. Vucic, Steve, Garth A. Nicholson, and Matthew C. Kiernan. “Cortical hyperexcitability may precede the onset of familial amyotrophic lateral sclerosis.” Brain 131.6 (2008): 1540-1550.
  6. Kanai, Kazuaki, et al. “Motor axonal excitability properties are strong predictors for survival in amyotrophic lateral sclerosis.” J Neurol Neurosurg Psychiatry 83.7 (2012): 734-738.
  7. Igaz, Lionel M., et al. “Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice.” The Journal of clinical investigation 121.2 (2011): 726-738.
  8. Saba, Luana, et al. “Modified age-dependent expression of NaV1. 6 in an ALS model correlates with motor cortex excitability alterations.” Neurobiology of disease 130 (2019): 104532.
  9. Weskamp, Kaitlin, et al. “Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS.” The Journal of clinical investigation 130.3 (2020).

 

 

 

Tags: amyotrophic lateral sclerosis, hyperexcitability, tdp-43

Posted on: 1 August 2020 , updated on: 31 October 2020

doi: https://doi.org/10.1242/prelights.23692

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Author's response

Marcus Dyer shared

1) Extranuclear TDP-43 is known to be involved in stress-granule formation and shows the propensity to form aggregates. Did the authors observe respective TDP-43 assemblies in layer V cortical neurons with TDP-43 mislocalization showing abnormal excitability?

 

We did not look directly for TDP-43 aggregation, though previous studies suggest in this model aggregates are generally quite rare, in less than 1% of cortical neurons (Igaz 2011). Our study was focusing upon determining if TDP-43 accumulating inappropriately would cause changes in neuronal excitability. It would be interesting in future studies to look at the protein forms of TDP-43 in the cytoplasm of the Layer V neurons and if there is any correlation to excitability phenotype burden

 

2) Using the same mouse model, it has previously been reported that TDP-43 mislocalization induced by the lack of the NLS results in a significant loss of neurons (9). However, in the motor cortex assessed in this study, no overt cell death was recognized. Do you have any explanation for this discrepancy?

 

Yes, previous studies in this mouse model have shown cell loss in the dentate gyrus (Igaz 2011) and in the motor cortex with NeuN (Alfieri 2014). We use CTIP2 as a marker for projection neurons out of the motor cortex as that is the population that we were particularly interested in with regards to ALS. So the discrepancy could be related to the population of cells under investigation. Further, we did not commence any experiments until we had fully backcrossed, and confirmed the congenic status, of this mouse onto the Bl6C57 background. The system that this model uses to drive TDP-43 expression (Tet off) has been reported to cause non-specific neurodegeneration if a mixed background is used (Han et al., 2012).

 

3) Did you observe any phenotypical changes in animals showing hyperexcitability?

 

Yes, we do observe a phenotype change. We did behavioural experiments when we first back crossed the mouse model in our lab. Some things we observe at 1 month of expression are limb clasping, severe lack of time on the rotarod as well as cognitive changes indicative of hyperexcitability.  We are still exploring this.

 

4) It is still under debate whether and to what extend depletion of TDP-43 from the nucleus or presence of TDP-43 in the cytoplasm contribute to TDP-43 toxicity. Does mislocalization of hTDP-43 affect TDP-43 levels in the nucleus in your model?

 

That is a good point. It has previously been shown in this model that there is both accumulation of TDP-43 in the cytoplasm and loss of endogenous nuclear TDP-43 (Igaz 2011). It would be really nice to show which one is most important for the progression of ALS, however, loss of nuclear TDP-43 and gain of cytoplasmic TDP-43 is very clinically relevant and perhaps most correctly models ALS in patients.

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