Toxic Loss & Gain of Function in a New TDP-43ΔNLS ALS Mouse Model

The SNc has an established role in motor control, making it a practical site for inducing and assessing motor dysfunction. Evidence indicates that phosphorylated TDP-43 (pTDP-43) pathology appears in the SNc at Braak stage 2, suggesting early involvement in ALS (Braak, 2013).

Extrapyramidal involvement in ALS can contribute to symptoms such as rigidity, postural instability, and balance problems (Desai, 1999; Pradat, 2009).

In this model, the AAV-hTDP43ΔNLS is controlled by a neuron-specific promoter. As shown in the photomicrograph, there is significant hTDP-43 expression in the targeted ipsilateral SNc and midbrain. For reference, an illustration with atlas labels for these brain regions is provided below.

This micrograph shows the cytoplasmic mislocalization of hTDP-43 resulting from the deletion of its nuclear localization signal (ΔNLS). The model was designed to replicate a hallmark feature of ALS pathology, where TDP-43 undergoes mislocalization from the nucleus to the cytoplasm, leading to the formation of cytoplasmic inclusions.

This microscopy image of the SNc in the targeted hemisphere shows hTDP-43 deposition within tyrosine hydroxylase (TH)-positive neurons. TH expressing cell bodies and processes are labeled in red, while hTDP-43 appears in green.

Colocalization of hTDP-43 with TH-positive neurons suggests a potential link between TDP-43 pathology and the motor deficits observed in this model, despite the absence of substantial dopaminergic cell loss. These findings suggest a functional impairment, rather than degeneration, of the SNc, likely driven by a toxic loss of function of TDP-43 in the nucleus.

Using our PERMITS™ quantitative analysis software, we have quantified the TH staining in the SNc. The plot below shows no significant difference in the ipsilateral hemisphere of the AAV-hTDP43 compared to the AAV-null (control) mice.

The boxed region highlights a marked increase in microglia, visualized by Iba-1 staining, within the targeted hemisphere compared to the contralateral side. This elevated density suggests a localized neuroinflammatory response to hTDP-43 accumulation.

The plot below shows the Iba-1 stain density in the ipsilateral hemisphere of the SNc.

Increased microglial density in the SNc and midbrain of the targeted hemisphere is localized in proximity to hTDP-43 accumulation, suggesting that the microglial response may be linked to the buildup of hTDP-43.

An increased density of GFAP-stained astrocytes is observed within the targeted hemisphere, particularly in the SNc and midbrain, as indicated by the boxed region. The plot below shows the GFAP stain density in the ipsilateral hemisphere of the SNc.

A higher density of GFAP-stained astrocytes is observed in regions adjacent to hTDP-43 accumulation.

Phosphorylated TDP-43 (pTDP-43) represents a pathological, disease-associated form of the protein,

A sparse distribution of pathological pTDP-43 aggregates is observed in the midbrain of the targeted, ipsilateral hemisphere at this stage of the disease progression.

In this high-magnification image, pTDP-43 is mislocalized to the cytoplasm. DAPI staining marks cell nuclei, while pTDP-43 shows well-defined cytoplasmic inclusions. Although pTDP-43 aggregates in the midbrain are sparse, mice nonetheless exhibited significant behavioral deficits, suggesting that even limited mislocalization of pTDP-43 in this region may be sufficient to impair normal functioning.

In a separate group of mice, a similar AAV-based approach was used to express hTDP43ΔNLS, this time through unilateral injection into the motor cortex.

In this microscopy image of the motor cortex, robust expression and mislocalization of pTDP-43 is evident, compared to the relatively sparse aggregation in the SNc or midbain. The increased pTDP-43 expression observed in the motor cortex suggests a region-specific vulnerability to TDP-43 pathology, which is particularly relevant given the well-established role of the motor cortex in ALS.

Mitochondrial dysfunction is a hallmark of several neurodegenerative diseases, including ALS. In this study using our AAV-based delivery of hTDP43-ΔNLS, ATP5A staining reveals punctate structures, consistent with dilated mitochondria in the motor cortex. This observation underscores the contribution of mitochondrial alterations to ALS pathophysiology and highlights the motor cortex as a particularly vulnerable region.

This novel AAV-hTDP43ΔNLS mouse model mirrors key pathological features of ALS, including the development of motor deficits and associated colocalization of hTDP-43 with TH-expressing neurons in the SNc. Importantly, hTDP-43 mislocalizes from the nucleus to the cytoplasm, resulting from the deletion of its nuclear localization signal (ΔNLS).

Dopaminergic cell death in the SNc is minimal, suggesting neuronal dysfunction, rather than neurodegeneration. Further, microglial and astrocyte densities in the SNc and midbrain are elevated, suggesting a link between TDP-43 accumulation and neuroinflammation.

This AAV-based model is well-suited for drug development and possesses distinct advantages, such as enabling region-specific targeting. Moreover, it can be rapidly established, making it an efficient model for screening novel treatment options targeting TDP-43-related pathology.

Further studies are planned to continue to investigate the range and progression of pathological changes in the model, and to further characterize the changes observed in motor cortex-injected mice. Our preliminary findings indicate a region-specific vulnerability of the motor cortex to pTDP-43 cytoplasmic aggregation and alterations of mitochondrial structure (and potentially function).

Please feel free to further explore the microscopy image in the viewer.

We would be happy to discuss this model and our characterization if you would like to Contact Us.