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Dong et al. Neuroimmunol Neuroinflammation 2018;5:5 I http://dx.doi.org/10.20517/2347-8659.2017.47 Page 7 of 10

[31]
misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells . In vitro,
immunocytochemistry and immunoprecipitation were used to demonstrate that TDP-43 or FUS-induced
misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding
[31]
of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion . While siRNA in recipient cells
and incubation of conditioned media with misfolded SOD1-specific antibodies could inhibit intercellular
[30]
transmission in vitro , intercellular spread of SOD1 misfolding is not accompanied by transmission of
TDP-43 or FUS pathology.

The spreading of pathological TDP-43
In vivo, TDP-43 protein is found in secreted exosomes from Neuro2a cells and primary neurons but not
[33]
astrocytes and microglia . The pathological TDP-43 protein aggregation and autophagy inhibition promote
[29]
exosomal secretion of TDP-43 . The levels of exosomal full length TDP-43 and C-terminal fragment
species are upregulated in the brain of ALS patients. If Neuro2a cells are exposed to the cerebrospinal
fluid of ALS patients, the deposition of intracellular pathological TDP-43 proteins would be take place.
In addition, Neuro2a cells can have TDP-43 deposition by regulating silent genes or inhibiting exocrine
secretion. Upregulation of exocrine secretion in the TDP-43 A315T mutant transgenic mice exacerbates the
[29]
disease process. Exosome secretion is considered a key pathway for clearance of pathological TDP-43 .

To study the potential propagation of TDP-43, a HEK293 cell culture model was used, which supports the
propagated misfolding of HuWtSOD1. It showed significant protein expression in cells transfected with
TDP-43 constructs, but no expression in the incubated cells, indicating that the conditioned media contains
no active residual lipofectamine reagent and that the transfection-encoded TDP-43 protein does not transmit
[31]
to recipient HEK293 cell cultures . Thus, pathological TDP-43 is cleared via the proteasome, which reduces
efficient clearance of mis-folded SOD1.

Recently, novel intracytoplasmic inclusions immunoreactive for phosphorylated transactivation response
TDP-43 (p-TDP 43) were found in anterior horn cells in a case of a sporadic amyotrophic lateral sclerosis
(sALS) patient. His spinal cord showed severe degeneration involving the anterior and lateral funiculi,
whereas the posterior funiculus was preserved. Most neurons in the anterior horn and Clarke’s column
were markedly lost, while some remaining anterior horn cells had round and densely eosinophilic or
[32]
amphophilic intracytoplasmic inclusions . They were immune-reactive for ubiquitin, p-TDP-43, cystatin C
[32]
and transferrin . On confocal laser microscopy, cystatin C was found to consistently surround p-TDP-43
within the inclusions. It was a key finding that these unique inclusions may have been formed under a
[33]
specific condition whereby p-TDP-43 and cystatin C interacted with each other .
Removal of pathological TDP-43
Although the tardbp gene mutation is uncommon, other gene mutations related to ALS can also lead
to abnormal intracellular TDP-43 levels [29,34] . Therefore, it is necessary to analyze the clearing pathway
of pathological TDP-43. The published study has reported such process not only involved the dynamic
[26]
transportation by exocrine secretion and small vesicles, but also by autophagy as well . With overcoming
the confounding effects of aggregation and toxicity, pathogenic mutations that significantly shorten
TDP-43 half-life were found in a single-cell optical method. A novel autophagic flux assay combined with an
in silico screen identified compounds that effectively stimulate autophagy in neurons though enhancement
of TDP-43 clearance and reduction of its mis-localization. On the other hand, the induction of autophagy
[26]
can improve scavenging ability of TDP-43, enhancing the primary neuronal survival capacity . TDP-
43 causes differential pathology in neuronal versus glial cells in the mouse brain by increasing the TDP-43
[26]
clearance which can slow down the progression of ALS .
The accumulation of intracellular TDP-43 is also related to the impairment of TDP-43 CTFs fragment
clearance. The autophagy mediated TDP-43 CTFs fragmentation is caused by the failure of the phagocytosis