TDP-43小鼠模型，肌萎缩侧索硬化症（ALS）

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.

This image shows a sagittal view of a Low Dox mouse brain immunostained for human TDP-43. Note the spatial pattern of cytoplasmic TDP-43 expression, with strong intensity in various brain regions, including the cerebral cortex, hippocampus, thalamus, hypothalamus, olfactory bulb, cerebellum, and brainstem.

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.

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.

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

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.

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).

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

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.

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.

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