TDP-43 Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

Q: Has disease-modification been shown in the TDP43 transgenic model?

Yes. Here is a nice example of a small molecule resulting in disease modification in this TDP43 mouse model: Young, P.R., DeDuck, K., Bedell, B.J. AIT-101 improves functional deficits in a human TDP-43 animal model of ALS. 22nd Annual Meeting of the Northeast ALS Consortium, 2023; doi:10.1002/mus.27969.

Q: What is a "nuclear localization signal"?

A nuclear localization signal (NLS) is typically a short peptide which facilitates the transport of a protein from the cytoplasm into the nucleus of a cell. A review can be found at doi:10.1186/s12964-021-00741-y.

Q: Are phosphorylated TDP43 aggregates seen in the TDP43 mice?

Yes. We have developed excellent immunohistochemistry (IHC) and immunofluorescence (IF) protocols that nicely demonstrate "punctate" p-TDP-43 (p409/410) aggregates in the cytoplasm of neurons without nuclear TDP-43 staining.

Q: What types of therapeutic agents has Biospective evaluated in this TDP43 transgenic model?

We tested a wide range of therapeutic agents, including antibodies, gene therapy, small molecules, antisense oligonucleotides, and peptides using a variety of different routes of administration in this TDP43 model.

Q: Can Biospective perform oral dosing in the TDP43 transgenic ALS model?

Yes. We routinely perform oral gavage of pharmacological agents in this TDP43 mouse model. We can also deliver via food or drinking water.

Q: How long are Biospective's studies involving the "Low Dox" mouse model of ALS?

Typical in-life study durations range from 3-12 weeks. We can work with you to define the optimal duration to assess disease progression and therapeutic effects based on your particular therapeutic agent.

Q: Are mice readily available for studies?

Yes. As a Preclinical Neuroscience CRO, we maintain a well-established breeding colony of the TDP43 ALS mice so that they are readily available for studies. We would be happy to discuss study timelines with you.

Our TDP-43 (TDP43; TARDBP) transgenic mice have cytoplasmic aggregates, motor dysfunction, neurodegeneration, neuroinflammation, and neuromuscular denervation.

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TDP-43 Model Overview

Cytoplasmic TDP-43 (or TDP43) aggregates are a hallmark of familial and sporadic ALS. While several transgenic (tg) mouse models of amyotrophic lateral sclerosis (ALS; also called motor neuron disease [MND]) with TDP-43 aggregation exist, they each have their respective strengths and weaknesses. Learn more about animal models of ALS in our Resource - ALS Mouse Models for Drug Development.

At Biospective, we use both the original and modified versions of the rNLS8 (or ΔNLS; delta NLS; dNLS) ALS mouse model of TDP-43 proteinopathy ("TDP-43 models"):

Original mouse model ("Off Dox"): rapidly progressing (weeks)
Biospective mouse model ("Low Dox"): slower progressing (months)

Significant advantages of these TDP-43 models for ALS researchers include:

Mislocalization of TDP-43 to the cytoplasm
Progressive motor deficits
Muscle weakness, denervation, and atrophy
Motor neuron degeneration & regional brain atrophy
Neuroinflammation
Brain, spinal cord, and neuromuscular junction (NMJ) pathology

The model time course is predictable and the measures of disease progression are highly reproducible, making it an excellent model for the evaluation of therapeutic agents in preclinical studies. Learn more in our Resource - TDP-43 ΔNLS (rNLS8) Mice for Drug Development.

TDP-43 Mice Generation

rNLS8 (NEFH-hTDP-43-ΔNLS) double transgenic ALS mice ("TDP43 mouse model") are generated by breeding mice having the NEFH-tTA transgene with mice having the tetO-hTDP-43-ΔNLS transgene. This TARDBP model was originally developed and reported by Walker et al. (Acta. Neuropathol., 130: 643-670, 2015). It is a model of amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND). It can also be used as a TDP-43 pathology model of frontotemporal dementia (FTD) or frontotemporal lobar degeneration (FTLD).

These TDP-43 transgenic mice are maintained on a Dox diet during breeding and the initial aging period (typically 5 to 12 weeks-of-age). The mice are then changed from a Dox diet to a standard diet ("Off Dox" model) or an alternate protocol developed by Biospective ("Low Dox" model) to allow for human TDP-43 expression. An interesting feature of this model is that pathologic and functional recovery can be achieved by putting mice back on a Dox diet.

Plot showing clasping score in different Dox conditions

Our Validated TDP-43 Transgenic Mice Measures

Body weight
Motor scoring (hindlimb clasping, tremor, grill agility, paralysis)
Grip strength test
In vivo muscle electrophysiology, including Compound Muscle Action Potential (CMAP) (see ALS Mouse Models & Spinal Motor Neurons)
Muscle atrophy measured by longitudinal, in vivo computerized tomography (CT) (see TDP-43 ΔNLS (rNLS8) Mice for ALS Drug Development)
Neurofilament light chain measures in plasma & CSF
MRI brain atrophy to measure neurodegeneration (see Brain Atrophy Analysis in Mouse Models of Neurodegeneration)
Immunohistochemistry & multiplex immunofluorescence

Learn more about the translatability of this model to human ALS.

Microscopy Images

The Interactive Image Viewer below allows you to explore an entire Multiplex Immunofluorescence tissue section from our TDP-43 transgenic mouse model.

You can pan around the image using the left mouse button. You can zoom in and out using the mouse/trackpad (up/down) or the + and - buttons in the upper left corner. You can toggle (on/off), change color, and adjust image settings for the channels in the Control Panel in the upper right corner.

We suggest using Full Screen Mode for the best interactive experience.

Multiplex Immunofluorescence of Brain Sections from the “Low Dox” TDP-43ΔNLS Mouse Model of ALS

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This Interactive Microscopy Image Story illustrates some of the interesting pathologic features of Biospective's "Low Dox" TDP-43ΔNLS mouse model.

This ALS model was specifically intended to have a slower disease progression compared to the original rNLS8 model, and to allow greater potential to detect disease-modifying therapeutic effects. We have found intriguing temporal dynamics of different pathologic aspects (e.g. neurodegeneration, astrogliosis, microgliosis) in this model.

Here, we explore the human TDP-43 expression in neurons and the neuroinflammatory response in a coronal brain section from this model.

This multiplex immunofluorescence (mIF) image was generated by immunostaining for hTDP-43, GFAP, Iba-1, and counterstained with the DAPI nuclear stain. Tissue sections were digitized using a high-throughput slide scanner and were processed using Biospective's PERMITS^TM software platform.

To navigate though this Image Story, you can use the arrows and/or the Table of Contents icon in the upper right corner of this panel.

https://opt003stagmediafiles.blob.core.windows.net/image/59d0970eaf9748f7a10f45935e285d2e

You can also interact with the microscopy image in the viewer on the right at any time to further explore this high-resolution data.

Human TDP-43 Expression

In this model, the human TDP-43ΔNLS transgene is under the control of the neurofilament heavy chain (NEFH) promoter (Walker, 2015). As such, expression is observed in neurons throughout the central nervous system (CNS).

As can be seen in this microscopy image, there is heterogeneity in the expression of human TDP-43 levels in neurons in different brain regions (e.g. cerebral cortex vs. caudate-putamen). For reference, an illustration with atlas labels for this brain level is provided below.

Mouse Brain Section (Bregma +0.75) with Neuroanatomy and Cortical Layer Labels

Cytoplasmic Mislocalization of hTDP-43

This mouse model was specifically designed to develop TDP-43 aggregates in the cytoplasm. The human TDP-43 has a defective nuclear localization signal (NLS) (Igaz, 2011). Walker and colleagues have also shown reduced expression levels of endogenous mouse TDP-43 in the nucleus as a result of cytoplasmic TDP-43 accumulation (Walker, 2015).

In this image from the motor cortex, note the high level of staining in the cytoplasm relative to the nucleus. The video below shows the hTDP-43 and DAPI toggled on/off to clearly see the spatial localization of hTDP-43.

Regional Cortical & Subcortical Astrogliosis

A prominent reactive astrogliosis is found in close proximity to degenerating motor neurons in ALS patients and animal models of ALS. While reactive astrogliosis in ALS is likely both primary and secondary to motor neuron degeneration, astrocytes are not simple bystanders and can influence the fate of motor neurons (Vargas, 2010).

This image shows the GFAP immunofluorescence staining. Astrogliosis is apparent in portions of the motor and somatosensory cortex, as well as the caudate-putamen.

Cortical Astrocytes in Laminar Pattern

The arrows highlight this laminar pattern in Layer 4 of the somatosensory cortex, while the box shows astrocytes in the motor cortex.

Astrocytes & TDP-43ΔNLS Expressing Neurons

In this image, one can readily appreciate the spatial relationship between the GFAP-stained astrocytes and the TDP-43 stained neurons.

We have found cortical atrophy in this model using in vivo anatomical MRI scans and advanced image processing & analysis methods.

Note the regional cortical thinning (green & yellow colors) in this animation, corresponding to regions of astrogliosis in the multiplex IF image. This multi-modality data suggests a regional (and potentially laminar) vulnerability of specific neuronal populations to mislocalized TDP-43.

Activated Microglia & TDP-43ΔNLS Model

Microglia appear to have central role in the pathologic and functional features in this ALS model. Examples of activated microglia with morphological changes (e.g. hypertrophic cell bodies, shorter processes) can be seen in this microscopy image from the somatosensory cortex.

Our team has developed advanced image processing tools that allow for analysis of microglial morphology, and we have been applying this technique to IHC & IF sections from various neurodegenerative disease models.

By leveraging our large dataset of IHC/IF images from TDP-43ΔNLS mice, we have identified a strong correlation between the density of non-ramified ("activated") microglia and the composite motor score (a combination of clasping, tremor, grill agility, hindlimb paralysis, and overall well-being scores).

An Image Showing the Relationship between Motor Scores and Microglia

Regional microglia morphological changes are highly correlated with the clinical composite (motor) scores (r=0.83).

Microglia & hTDP-43 Expressing Neurons

Spiller et al. (Spiller, 2018) found a shift in morphology from resting/homeostatic (ramified) to activated microglia during the disease “recovery” after expression of pathological TDP-43 was halted. These reactive microglia selectively cleared the neuronal hTDP-43 and there was a concomitant functional recovery. We have also observed this recovery of motor function in this model.

An Image showing a Plot of Hindlimb Clasping

When Spiller and colleagues (Spiller, 2018) blocked microgliosis with the CSF1R/c-kit inhibitor, PLX3397, during the early recovery phase, the mice failed to regain full motor function, revealing a neuroprotective role of microglia in this model.

This ability to facilitate clearance of cytoplasmic TDP-43 may be mediated via microglial-neuronal interactions (Cserép, 2021). Neuroinflammatory interactions between microglia and neurons occur at both synapses and the soma, with synaptic interactions regulating pruning, plasticity, and network synchronization. When dysfunctional, these interactions can lead to pathological synapse elimination and neurodegeneration (Clark, 2012; Pascual, 2012; Hong, 2016). Soma interactions, recently identified as critical for monitoring neuronal health, can provide neuroprotection or, when dysregulated, contribute to chronic inflammation and neuronal death (Salter, 2017; Cserép, 2021).

The arrow on the microscopy image from the motor cortex indicates potential contact points between the neuron soma and microglial processes. Our team is actively analyzing these glial-neuronal interactions and their roles in disease pathogenesis.

References

Clark, A.K., Malcangio, M. Microglial signalling mechanisms: cathepsins and fractalkine. Exp. Neurol., 234: 283–292, 2012; doi: 10.1016/J.EXPNEUROL.2011.09.012

Cserép, C., Pósfai, B., Dénes, A. Shaping neuronal fate: functional heterogeneity of direct microglia-neuron interactions. Neuron, 109: 222-240, 2021; doi: 10.1016/j.neuron.2020.11.007

Hong, S., Beja-Glasser, V.F., Nfonoyim, B.M., Frouin, A., Li, S., Ramakrishnan, S., Merry, K.M., Shi, Q., Rosenthal, A., Barres, B.A., Lemere, C.A., Selkoe, D.J., Stevens, B. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science, 352: 712–716, 2016; doi: 10.1126/SCIENCE.AAD8373

Igaz, L.M., Kwong, L.K., Lee, E.B., Chen-Plotkin, A., Swanson, E., Unger, T., Malunda, J., Xu, Y., Winton, M.J., Trojanowski, J.Q., Lee, V.M.-Y.. Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J. Clin. Invest., 121: 726–738, 2011; doi: 10.1172/jci44867

Pascual, O., Achour, S. Ben, Rostaing, P., Triller, A., Bessis, A. Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission. Proc. Natl. Acad. Sci. USA, 109: 2012; doi: 10.1073/PNAS.1111098109

Salter, M.W., Stevens, B. Microglia emerge as central players in brain disease. Nat. Med., 23: 1018–1027, 2017; doi: 10.1038/NM.4397

Spiller, K. J., Restrepo, C. R., Khan, T., Dominique, M. A., Fang, T. C., Canter, R. G., Roberts, C. J., Miller, K. R., Ransohoff, R. M., Trojanowski, J. Q., Lee, V. M. Y. Microglia-mediated recovery from ALS-relevant motor neuron degeneration in a mouse model of TDP-43 proteinopathy. Nat. Neurosci., 21: 329–340, 2018; doi: 10.1038/s41593-018-0083-7

Vargas, M.R., Johnson, J.A. Astrogliosis in amyotrophic lateral sclerosis: role and therapeutic potential of astrocytes. Neurotherapeutics, 7: 471-81, 2010; doi: 10.1016/j.nurt.2010.05.012

Walker, A.K., Spiller, K.J., Ge, G., Zheng, A., Xu, Y., Zhou, M., Tripathy, K., Kwong, L.K., Trojanowski, J.Q., Lee, V.M.-Y. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol., 130: 643-660, 2015; doi: 10.1007/s00401-015-1460-x

Table of Contents

Section: Coronal Brain

Zoom level

Channels

Nuclei (DAPI)

Microglia (Iba-1)

Astrocytes (GFAP)

Human TDP-43

Learn more about our characterization of this model, our validated measures, and our Preclinical Neuroscience CRO services.

FAQs

Has disease-modification been shown in the TDP43 transgenic model?

What is a "nuclear localization signal"?

What is the advantage of Biospective's "Low Dox" mouse model over the conventional model?

The standard rNLS8 (or delta NLS; ΔNLS; dNLS) mouse model is a rapidly progressing model of ALS (also known as motor neuron disease; MND). While this model is very useful, we have found that most of our Sponsors want a less severe, slower progressing model to increase the opportunity to observe a drug effect. The Low Dox TDP-43 mice show a similar phenotype to the standard model, including neurodegeneration, but evolves over a longer period of time.

Are phosphorylated TDP43 aggregates seen in the TDP43 mice?

What types of therapeutic agents has Biospective evaluated in this TDP43 transgenic model?

Can Biospective perform oral dosing in the TDP43 transgenic ALS model?

How long are Biospective's studies involving the "Low Dox" mouse model of ALS?

Are mice readily available for studies?

TDP-43 Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

Our TDP-43 (TDP43; TARDBP) transgenic mice have cytoplasmic aggregates, motor dysfunction, neurodegeneration, neuroinflammation, and neuromuscular denervation.

TDP-43 Model Overview

TDP-43 Mice Generation

Our Validated TDP-43 Transgenic Mice Measures

Microscopy Images

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