Research Models

NEFH-tTA x hTDP-43ΔNLS

Synonyms: rNLS8, regulatable NLS

Species: Mouse
Genes: TARDBP
Modification: TARDBP: Transgenic
Disease Relevance: Amyotrophic Lateral Sclerosis, Frontotemporal Dementia
Strain Name: B6;C3-Tg(NEFH-tTA)8Vle Tg(tetO-TARDBP*)4Vle/J
Genetic Background: NEFH-tTA mice and tetO-hTDP-43ΔNLS line 4 mice were maintained on a mixed C57BL/6J x C3HeJ background.
Availability: Available through The Jackson Lab as single transgenics: Stock# 025397 and Stock# 014650; Live. See also double transgenic Stock# 028412; Live

Summary

Virginia Lee, John Trojanowski, and colleagues at the University of Pennsylvania developed this bigenic model which expresses a regulatable form of human TDP-43 lacking a nuclear localization signal (NLS) (Walker et al., 2015). Following transgene activation, the mice quickly accumulate cytoplasmic insoluble TDP-43 in neurons of the brain and spinal cord. In addition, these mice develop other features of ALS, including motor deficits, denervation of neuromuscular junctions, motor neuron loss, and premature death. The expression of human TDP-43 can be suppressed by administration of the tetracycline analog doxycycline (dox). This allows for transgene suppression during critical developmental periods (e.g., prenatally and five weeks postnatally) and enables researchers to investigate whether/and to what extent phenotypes are reversible following transgene suppression later in life.

In this bigenic model, one parental line contains a transgene encoding the tetracycline activator protein (tTA) under the control of the neurofilament heavy chain (NEFH) promoter. The resulting tTA protein drives expression of human TDP-43 resulting in widespread expression in neurons of the brain and spinal cord, as early as one week off dox. Expression was reportedly minimal/absent in peripheral tissues, including the spleen, kidney, and livers. Notably, in layer V of the motor cortex, greater than 95 percent of NeuN-positive neurons co-labeled with human TDP-43 after one week of transgene de-repression. In addition to the expected neuronal expression, 5 to 10 percent of GFAP-positive astrocytes were also positive for human TDP-43.

TDP-43 expression in rNLS8 mice is robust by two to four weeks after de-repression. Total TDP-43 protein levels in the brain were 10-fold higher than levels in non-transgenic littermates, with a concomitant 50 percent decrease in endogenous mouse TDP-43.

rNLS8 mice develop TDP-43 cytoplasmic inclusions at a young age; rare inclusions appear as early as one week after transgene activation. Inclusions become widespread throughout the brain, including the motor cortex, as well as the visual, entorhinal and somatosensory cortices, cerebellum, and hippocampus. Ubiquitin-positive inclusions are also observed.  In the spinal cord, TDP-43 positive are comparatively rare, reported in 2 percent of spinal cord motor neurons at end stage.

Neuronal degeneration in the brain is detectable beginning at four weeks off dox, as reflected by a decrease in cortical thickness. In parallel, astrogliosis develops in many brain regions including layer V of the motor cortex. By end stage, the mice have significantly smaller brains than their littermate controls. Motor neuron loss in the spinal cord develops slightly later, with loss of ventral horn motor neurons evident by six weeks off dox. 

Quantification of neuromuscular junctions in the hindlimb revealed early axonal die-back and denervation, about four weeks off dox. This is approximately two weeks prior to detectable loss of lower motor neurons. Muscle atrophy follows next, as indicated by reduced mass of the hindlimb muscles, tibialis anterior, and gastrocnemius.

These mice develop a variety of motor impairments, including an early onset hindlimb-clasping phenotype and a fine tremor in their forelimbs and/or hindlimbs. They also develop a progressive loss of grip strength as measured by the wire-hang test and a progressive decline in coordinated movement and balance as measured by the accelerating Rotarod.

Body mass peaks around 7 weeks of age (i.e., two weeks off dox) and then progressively decreases in both males and females. Excessive loss of body weight, (defined as greater than 30 percent loss from peak weight) was the primary factor defining end-stage disease. Median survival was 10.3 weeks off dox (i.e., about 15.3 weeks of age).

Males and females exhibited similar phenotypes and were grouped together for analyses.

TDP-43 pathology and functional deficits were found to be largely reversible even after neurodegeneration was underway. In a paradigm involving six weeks of transgene activation followed by suppression for two-plus weeks, cytoplasmic TDP-43 cleared and endogenous mouse TDP-43 returned to the nucleus. Rapid functional improvements were observed, including partial recovery of hindlimb clasping phenotype, and improved performance on the Rotarod and wire-hang tests. Further motor neuron loss in the spinal cord was prevented. The mice gained weight rapidly and their lifespan was extended by at least five to eight months.

Availability

Both parental lines are distributed through The Jackson Lab. The double transgenic (Stock# 028412) is created by breeding NEFH-tTA line 8 (Stock# 025397) with TDP-43ΔNLS transgenics (tetO-hTDP-43-ΔNLS line 4) Stock# 014650. The Jackson Lab does not distribute the double transgenic; see their website for the most current availability information.

Related Strains

These mice represent a "second-generation" model of regulatable TDP-43 lacking a NLS. The team at U. Penn had previously generated a bigenic model called hTDP-43ΔNLS, which expresses the same TDP-43 responder transgene. In the earlier model tTA was driven by CAMKIIα rather than NEFH, resulting in preferential expression in forebrain neurons (Igaz et al., 2011).

NEFH-hTDP-43-WT - This is a regulatable contol line expressing wildtype TARDP which is also available through The Jackson Lab Stock# 028413. This line is created by breeding NEFH-tTA line 8 (Stock# 025397) with TRE-promoter-driven human TDP-43 wildtype transgenic mice (tetO-hTDP-43-WT line 12; Stock# 016841). On a mixed C57BL/6J x C3HeJ F1 background, mice hemizygous for both NEFH-tTA and tetO-hTDP-43-WT display nuclear human TDP-43 in the brain and spinal cord. They do not develop cytoplasmic TDP-43 pathology.

Phenotype Characterization

When visualized, these models will distributed over a 18 month timeline demarcated at the following intervals: 1mo, 3mo, 6mo, 9mo, 12mo, 15mo, 18mo+.

Absent

No Data

Cortical Neuron Loss

Decreased cortical thickness indicative of neuronal degeneration beginning at four weeks off dox. By end stage, rNLS8 mice had significantly smaller brains than non-Tg littermates.

Lower Motor Neuron Loss

rNLS8 lost motor neurons in the lumbar spinal cord by six weeks off dox.

Cytoplasmic Inclusions

Cytoplasmic inclusions of TDP-43 occur as early as one week off dox in neurons in the brain. Inclusions accumulate over time and are present in many brain regions, including the motor cortex. TDP-43 inclusions are relatively rare in the spinal cord. Ubiquitin-positive inclusions are also seen.

Gliosis

Astrogliosis develops in many brain regions, including layer V of the motor cortex.

NMJ Abnormalities

Denervation of the hindlimb muscle tibialis anterior was detectable by four weeks off dox, that is, two weeks prior to detectable loss of lower motor neurons.

Muscle Atrophy

At end-stage, rNLS8 mice exhibit gross muscle atrophy of the hindlimb muscles tibialis anterior and gastrocnemius.

Motor Impairment

rNLS8 mice develop a variety of motor impairments, starting with a deficit in hindlimb clasping and a fine tremor in the forelimb and/or hindlimb. They also develop progressive loss of grip strength (as measured by the wire-hang test) and a progressive decline in coordinated movement and balance (as measured by the accelerating Rotarod).

Body Weight

Body mass peaked at approximately 7 weeks of age (i.e., two weeks off dox) and then progressively dropped. Excessive loss of body weight (>30% decrease from peak weight) often defined end-stage.

Premature Death

rNLS8 mice die prematurely. They reach end-stage 8-18 weeks off dox, with a median survival of 10.3 weeks off dox.

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References

Research Models Citations

  1. hTDP-43ΔNLS

Paper Citations

  1. . Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol. 2015 Nov;130(5):643-60. Epub 2015 Jul 22 PubMed.
  2. . Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest. 2011 Feb;121(2):726-38. Epub 2011 Jan 4 PubMed.

External Citations

  1. Stock# 028412
  2. Stock# 025397
  3. Stock# 014650
  4. The Jackson Lab Stock# 028413.
  5. Stock# 016841
  6. Stock# 025397
  7. Stock# 014650;
  8. Stock# 028412

Further Reading