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What is the TDP-43 ΔNLS transgenic mouse model?

The TDP-43 ΔNLS model, also known as deltaNLS, delta NLS, hTDP-43ΔNLS, hTDP-43DeltaNLS, NEFH-hTDP-43ΔNLS, dNLS, TDP43 NLS, rNLS, and rNLS8, is a transgenic mouse originally reported by Walker and colleagues in 2015. The key, unique characteristic of this model is that the expressed human TDP-43 protein has a defective nuclear localization signal (NLS) that prevents translocation to the nucleus (where it would normally localize and function). The resulting accumulation in the neuronal cytoplasm leads to the formation of aggregates, including phosphorylated TDP-43 aggregates, similar to those observed in ALS patients.

In this model, the expression of hTDP-43ΔNLS is controlled by the neurofilament heavy (NEFH) promoter, thereby limiting it to neurons. In a predecessor model, reported by Igaz and colleagues, the expression was under the Camk2a promoter, which led to accumulated cytoplasmic TDP-43 in the brain, but with minimal pathological TDP-43 aggregates and lack of an ALS-like phenotype. The expression is "regulatable" (hence "rNLS") by the tetracycline analog, doxycycline (Dox). The rNLS8 double transgenic line, with expression of  hTDP-43ΔNLS in both the brain and spinal cord, is generated by breeding the individual transgenic lines: NEFH-tTA (Line 8; NEFH promoter directing tetracycline-controlled transactivator protein [tTA] expression) and tetO-hTDP-43ΔNLS (Line 4; tetracycline operator promoter [tetO] directing the expression of hTDP-43ΔNLS). The double transgenic mice are maintained on a Dox diet during breeding and the first several weeks-of-life (typically 5-12 weeks-of-age) to suppress the expression of hTDP-43ΔNLS. Disease induction is initiated by switching to standard chow (the "Off Dox" model). Note that mice maintained on the Dox diet ("On Dox" mice) can serve as a control group as they do not show signs of disease.

A key feature of this model is the ability to obtain a pathologic and functional recovery by reinstating the Dox diet. The halting and reversibility of the disease progression in this model demonstrates that disease modification is possible via mediation of TDP-43 expression and pathological aggregates, thereby making it an attractive model for ALS drug development.

The original ΔNLS model is rapidly progressing with weight loss and motor symptoms typically appearing within approximately one week from the removal of Dox to death within several weeks. As an alternative that is better suited for most therapeutic efficacy studies, our team developed a slower progressing version using an alternative Dox protocol. In our "Low Dox" model,  a similar phenotype is produced, but evolves over a longer period of time with mice surviving to at least 3 months after model induction.

What are key features of human ALS that are present in TDP-43 ΔNLS mice?

ALS is a fatal neurodegenerative disease that affects numerous systems in humans, including the central nervous system (CNS), peripheral nervous system (PNS), autonomic nervous system (ANS), muscle, and immune system. As summarized in the infographic below, the ΔNLS model has been demonstrated to recapitulate a number of these features, with others (e.g. ANS and immune system) still to be explored.

Multi-system effects of ALS in a mouse model

TDP-43 ΔNLS mice demonstrate multi-system involvement, including brain, spinal cord, neuromuscular junctions (NMJ), and muscles, thereby making it a good model for evaluation of putative therapeutic agents for ALS.

In the brain, ΔNLS mice show cytoplasmic TDP-43 aggregates (including phosphorylated TDP-43), neurodegeneration, impaired glymphatic function, neuroinflammation (microgliosis and astrogliosis), and a proteomic signature that correlates with that of post-mortem human brain tissue from patients with TDP-43 proteinopathies.

Two MRI images with one showing bright CSF

Anatomical MRI from control (left) and ΔNLS mice off Dox for 3 weeks (right). Note the bright signal in specific regions showing brain atrophy in the ΔNLS mice. 

In terms of neuroinflammation, microglia play an intriguing role in this model. Spiller et al. found that after TDP-43 expression was halted by putting mice back on Dox, microglia transiently proliferated and changed their morphology and gene expression, facilitating the clearance of the cytoplasmic TDP-43. When microglia were depleted, the recovery was attenuated. Hunter et al. performed a transcriptomic analysis during the progression and recovery phases, and found that differentially expressed genes were associated with chemotaxis, phagocytosis, inflammation, and production of neuroprotective factors. Swanson et al. found that microglia are phagocytic at early stages of the disease and transition to a dysfunctional state at later stages, and that these functional states are driven by phosphorylated TDP-43 aggregation. 

In the spinal cord, Spiller et al. found that slow motor neurons are resilient while fast fatigable motor neurons are lost, and axonal dieback occurs first from fast-twitch muscle fibers, while slow-twitch fibers remain innervated. These findings in the mouse model mirror the selective patterns of motor neuron degeneration in human disease.

Walker et al. reported significant early and progressive neuromuscular junction (NMJ) denervation. Hur et al. demonstrated that motor neuron identity is responsible for the susceptibility of NMJs to TDP-43 pathology, and that slow motor neurons can drive the recovery of motor systems as a function of their resilience to TDP-43 associated neurodegeneration. Several papers, including Spiller et al., have reported reduction of compound muscle action potential (CMAP) amplitude.

In terms of muscle involvement, muscle weakness, grouped fiber atrophy and centralized nuclei have been found in muscle fibers, and loss of muscle mass in the gastrocnemius and tibialis anterior muscles have been reported by Walker and colleagues. Tsitkanou et al. found hTDP-43 cytoplasmic accumulation of TDP-43 and oligo A11 β-amyloid in denervated muscle fibers in the tibialis anterior, quadriceps, and diaphragm muscles. They also found atrophy of the tibialis anterior, quadriceps, and gastrocnemius muscles, an upregulation of markers of myogenic and neuromuscular junction (NMJ) stress, impaired motor function and muscle force production, and dysregulation of several proteins related to mitochondrial complexes.

Our group has used non-invasive computed tomography (CT) image to demonstrate selective muscle atrophy, which most significantly affects the hindlimb gastrocnemius muscle, in our "Low Dox" mouse model.

CT image with segmentation of the hindlimb muscles from Control (On Dox) and Low Dox mice

CT image with segmentation of the hindlimb muscles from Control (On Dox) and "Low Dox" TDP-43 ΔNLS mice; medial gastrocnemius = blue; lateral gastrocnemius = yellow; posterior tibialis = purple; anterior tibialis= pink; soleus = magenta. 

3D volume rendering of the medial (blue) and lateral (yellow) gastrocnemius muscles from Control and "Low Dox" TDP-43 ΔNLS mice. 

What measures have been used to assess therapeutic efficacy in the TDP-43 ΔNLS ALS mouse model?

Several groups have shown disease modification via therapeutic intervention in this mouse model. Young et al. have demonstrated the ability of a small molecule PIKfyve inhibitor, AIT-101 (INN: apilimod, aka LAM-002A), to decrease the loss of body weight, reduce motor deficits (including hindlimb clasping, hindlimb paralysis, and grill agility), along with accompanying decreases in plasma and CSF neurofilament light (NfL) levels, TDP-43 aggregates (by IHC), and neuroinflammation (by GFAP IHC) in Biospective's Low Dox version of the rNLS8 model.

Stomakhina et al. found reduction in plasma and CSF neurofilament light (NfL) levels, as well as reduction in acid sphingomyelinase (ASM) activity in the caudal cortex in rNLS8 mice treated with VRG50304 over a 28 day period. Droppelmann et al. found that intracerebroventricular injections of AAV9/NF242 N-terminal fragment of rho guanine nucleotide exchange factor [RGNEF]) in the rNLS8 model improved lifespan and motor phenotype (hindlimb clasping, open field, gait [CatWalk]), and decreased neuroinflammation markers (GFAP and Iba-1).

Our team would be happy to answer any questions about the TDP-43 ΔNLS ALS mouse model or provide specific information about the models that we use for therapeutic efficacy studies.

FAQs

How is CT muscle atrophy measured?

A high-resolution CT image of both hindlimbs is acquired. At Biospective, we have developed a fully-automated "pipeline" for quantitative image analysis. The CT images are first preprocessed to remove the acquisition bed from the scans. Each preprocessed image is then registered to a population average mouse template using a combination of linear and non-linear transformations. These transformations are inverted and are used to map an atlas defining the various leg muscles on the mouse template back onto the individual CT images to obtain native muscle volumes.


Can brain imaging be used to assess therapeutic efficacy in the ΔNLS mouse model?

Yes. Preclinical MRI is a powerful tool to assess brain atrophy in the ΔNLS model. Non-invasive anatomical MR imaging allows for longitudinal measures of regional brain volume and cortical thickness, and can serve as a sensitive, clinically-translational biomarker of potential disease modification via therapeutic intervention.


What is a reasonable sample size for a therapeutic efficacy study in ΔNLS mice?

In our studies using the Low Dox model, we typically include 10-15 mice per group.


How often is motor function assessed in therapeutic efficacy studies?

We typically measure hindlimb clasping, tremor, grill agility, and hindlimb paralysis 3 times per week for the duration of the study. Other measures (e.g. grip strength) can be measured at a few time points (e.g. baseline, 4 weeks, and 8 weeks).


What is the rNLS8 model?

The "regulatable" NLS (rNLS; rNLS8) model is the same as the TDP-43 ΔNLS model. It is the double transgenic mouse originally reported by Walker and colleagues in 2015. It is considered regulatable since the expression of the TDP-43 ΔNLS transgene (under the NEFH promoter) can be controlled via the administration of the tetracycline-analog doxycycline (Dox). The TDP-43 ΔNLS transgene expression is suppressed during administration of a high dose of Dox.


Does the TDP-43 ΔNLS mouse model show cytoplasmic aggregates?

Yes, the cytoplasmic mis-localization of TDP-43 and formation of aggregated TDP-43 (including phosphorylated aggregates) in the neuronal cytoplasm is a hallmark of this model resulting from the defective nuclear localization signal (NLS). 


References

Droppelmann, C.A., Campos-Melo, D., Noches, V., McLellan, C., Szabla, R., Lyons, R.A., Amzil, H., Withers, B., Sonkar, K.S., Simon, A., Buratti, E., Junop, M., Kramer, J.M., Strong, M.J. Mitigation of TDP-43 induced toxic phenotype by expression of RGNEF N-terminal fragment in ALS models. bioRxiv, 2023.09.29.560207, 2023; doi: 10.1101/2023.09.29.560207

Hunter, M., Spiller, K.J., Dominique, M.A., Xu, H., Hunter, F.W., Fang, T.C., Canter, R.G., Roberts, C.J., Ransohoff, R.M., Trojanowski, J.Q., Lee, V.M.-Y. Microglial transcriptome analysis in the rNLS8 mouse model of TDP-43 proteinopathy reveals discrete expression profiles associated with neurodegenerative progression and recovery. Acta. Neuropathol. Commun., 9: 140, 2021; doi: 10.1186/s40478-021-01239-x

Hur, S.W., Hunter, M., Dominique, M.A., Farag, M., Cotton-Samuel, D., Khan, T., Trojanowski, J.Q., Spiller, K.J., Lee, V.M.-Y. Slow motor neurons resist pathological TDP-43 and mediate motor recovery in the rNLS8 model of amyotrophic lateral sclerosis. Acta. Neuropathol. Commun., 10: 74, 2022; doi: 10.1186/s40478-022-01373-0

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

San Gil, R., Pascovici, D., Venurato, J., Brown-Wright, H., Mehta, P., Madrid San Martin, L., Wu, J., Luan, W., Kit Chui, Y., Bademosi, A.T., Swaminathan, S., Naidoo, S., Berning, B.A., Wright, A.L., Keating, S.S., Curtis, M.A., Faull, R.L.M., Lee, J.D., Ngo, S.T., Lee, A., Morsch, M., Chung, R.S., Scotter, E., Lisowski, L., Mirzaei, M., Walker, A.K. A transient protein folding response targets aggregation in the early phase of TDP-43=mediated neurodegeneration. Nat. Comm., 15: 1508, 2024; doi: 10.1038/s41467-024-45646-9

Spiller, K.J., Cheung, C.J., Restrepo, C.R., Kwong, L.K., Stieber, A.M., Trojanowski, J.Q., Lee, V.M.-Y. Selective motor neuron resistance and recovery in a new inducible mouse model of TDP-43 proteinopathy. J. Neurosci., 36: 7707-7717, 2016; doi: 10.1523/JNEUROSCI.1457-16.2016   

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 ina  mouse model of TDP-43 proteinopathy. Nat. Neurosci., 21: 329-340, 2018; doi: 10.1038/s41593-018-0083-7   

Stomakhina, E., Kim, G.,, Choi, I., Kopec, B., Zhang, N., Batia, L. Rescue of ALS relevant biomarkers by VRG50304 in rNLS8 mouse model. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 22(sup2): 107, 2023; doi: 10.1080/21678421.2021.1985792
 
Swanson, M.E.V., Mrkela, M., Murray, H.C., Cao, M.C., Turner, C., Curtis, M.A., Faull, R.L.M., Walker, A.K., Scotter, E.L. Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain. Acta. Neuropathol. Commun., 11: 69, 2023; doi: 10.1186/s40478-023-01561-6
 
Tsitkanou, S., Della Gatta, P.A., Abbott, G., Wallace, M.A., Lindsay, A., Gerlinger-Romero, F., Walker, A.K., Foletta, V.C., Russell, A.P. miR-23a suppression accelerates decline in the rNLS8 mouse model of TDP-43 proteinopathy. Acta. Neurobiol. Dis., 162: 105559, 2022; doi: 10.1016/j.nbd.2021.105559
 
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
 
Young, P.R., DeDuck, K., Bedell, B.J. AIT-101 improves functional deficits in a human TDP-43 animal model of ALS. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 24(sup1): 129, 2023; doi: 10.1080/21678421.2023.2260194
 
Zamani, A., Walker, A.K., Rollo, B., Ayers, K.L, Farah, R, O'Brien, T.J., Wright, D.K. Impaired glymphatic function in early stages of disease in a TDP-43 mouse model of amyotrophic lateral sclerosis. Transl. Neurodegen., 11: 17, 2022; doi: 10.1186/s40035-022-00291-4
 
Zamani, A., Walker, A.K., Rollo, B., Ayers, K.L., Farah, R., O'Brien, T.J., Wright, D.K. Early and progressive dysfunction revealed by in vivo neurite imaging in the rNLS8 TDP-43 mouse model of ALS. Neuroimage Clin., 34: 103016, 2022; doi: 10.1016/j.nicl.2022.103016


Keywords

Amyotrophic Lateral Sclerosis (ALS): also known as Lou Gehrig's disease, it is the most common form of motor neuron disease (MND) and affects the upper and lower motor neurons. This fatal neuromuscular disease is characterized by progressive weakness of the muscles required to move, speak, eat, and breathe.

Brain Atrophy: reduction in volume or thickness of the entire brain or regions of the brain.

Computed Tomography (CT): a non-invasive imaging modality that uses x-rays at multiple different angles to generate 3D images.

Compound Muscle Action Potential (CMAP): summed action potentials of all stimulated motor endplates. 

Doxycycline (Dox): a tetracycline analog that is used to regulate gene expression using a Tet-On or Tet-Off system. 

Hindlimb Clasping: one or both hindlimbs are retracted towards the abdomen when a mouse is suspended by the tail.

Low Dox Model: a variation of the standard ΔNLS model using a protocol developed by Biospective to generate a less severe, slower progressing phenotype.

Magnetic Resonance Imaging (MRI): a non-invasive imaging modality that uses magnetic fields and radiofrequency (RF) pulses to generate images.

Muscle Atrophy: reduction in volume or thickness of skeletal muscle. 

Nuclear Localization Signal (NLS): a short peptide which facilitates the transport of a protein from the cytoplasm into the nucleus of a cell.

Neuromuscular Diseases: disorders that affect the nerves that control voluntary muscles and the nerves that communicate sensory information to the brain.

Neuromuscular Junction (NMJ): a synaptic connection between the terminal end of a motor nerve and a muscle.

Neurodegeneration: a complex, multifactorial process resulting in the loss of neurons. 

Neurofilament Light (NfL; NF-L): one of four subunits of neurofilaments, which are proteins found in neurons that provide structure and shape; the neurofilament light level in blood and CSF can serve as marker of neuro-axonal damage.

Transactive response DNA binding protein of 43 kDa (TDP-43):  a highly conserved nuclear RNA/DNA-binding protein encoded by the TARDBP gene involved in the regulation of RNA processing.

Translational Biomarker: a robust indicator of a biological state or process that is measurable in both animal models and humans.


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