TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic ALS are low frequency and widely distributed in the lower motor neuron columns independent of disease spread

Abstract

Ubiqitinated and TDP-43 immunoreactive cytoplasmic aggregates are hallmark features of ALS molecular pathology. Since clinically most ALS begins focally and advances contiguously, it is important to characterize their distribution. Our objective was to determine the extent and distribution of TDP-43 immunoreactive aggregates in the lower motor neuron columns as a function of disease onset, and to correlate ubiquitinated with TDP-43 aggregates in the lumbar region. We examined TDP-43 cytoplasmic aggregates at four separate neuraxis levels - hypoglossal nucleus and cervical, thoracic, and lumbar anterior horns - in five controls and 20 sporadic ALS nervous systems from patients whose disease began in various sites, i.e. five bulbar, five arm, five trunk, and five leg onsets. We correlated ubiquitinated to TDP-43 aggregates on adjacent histological sections for the lumbar regions. We found that TDP-43 cytoplasmic aggregates are seen in about 8% of motor neurons but there is marked variability between nervous systems, ranging from 0.4% to 20.6%. The aggregates are uniformly distributed within individual nervous systems. There is no obvious correlation between site of disease onset and rate of spread. Almost all ubiquitinated aggregates correlate to TDP-43 aggregates. Thus, TDP-43 immunoreactive cytoplasmic aggregates have a low overall average frequency that does not correlate with either disease course or clinical spread and is the prime ubiquitinated protein.

Figure 1.

Morphological categories of TDP-43 immunoreactive cytoplasmic aggregates in ALS lower motor neurons. (A) Skeins: these appear as multiple string-like TDP-43-positive aggregates (arrow head). (B) Dense round inclusions: these appear as dense, round TDP-43-positive aggregates (arrow head). (C–D) Overlaps: these appear as an intermediate between skeins and dense round inclusions. In C, the TDP-43-positive aggregate appears similar to a dense round inclusion but has diffuse aggregation emerging from the inclusion body (arrow head). In D, morphology is similar to a skein, but the string-like deposits are locally condensed and form a large, dense deposit (arrow head). Note that the aggregates are seen in what otherwise appear to be morphologically healthy neurons. (Scale bar = 10μm).

Figure 2.

Bar graph showing the percentage of TDP-43 immunoreactive cytoplasmic aggregates at the cervical, thoracic, and lumbar spinal cord levels and the medulla for all 25 nervous systems. TDP-43 aggregates show no statistical correlation between frequency and anatomical distance from disease onset, and are not indexed to topographic advance of disease. Bar graph shows composite data.

Figure 3.

Correlation between ubiquitinated and TDP-43 immunoreactive cytoplasmic aggregates in ALS lower motor neurons. (A) Ubiquitinated aggregates in three separate motor neurons. (B) TDP-43 immunoreactive aggregates in the adjacent section showing corresponding changes (respective arrows). The morphological appearances are similar between ubiquitinated and TDP-43 immunostaining; inlays show respective magnified images (arrowhead).