C9ALS cases show different patterns of p62-positive inclusion pathological dependency when compared to sALS cases pathological dependency when compared to sALS cases

It was next sought to determine whether there was a relationship between the levels of p62-positive inclusion pathology shown within CNS regions in both C9ALS and sALS cases. Firstly, the relationship between neuronal and glial inclusions within the three CNS regions (spinal cord, motor cortex and anterior frontal cortex) was studied using Kendall correlation tests. The results of this study are presented in table 3.4 and figures 3.2 and 3.3.

Analysis of the spinal cord inclusion counts in C9ALS cases consistently revealed positive correlations between neuronal and glial inclusion pathology and between both anterior horn and corticospinal tract glial inclusion counts (figure 3.2A-C).

However, sALS cases only showed a significant relationship between grey matter and white matter glial cell counts, but no relationship was found between motor neurone and glial inclusions (figure 3.3A-C).

140 Evaluation of the motor cortex glial inclusion counts again showed a significant correlation between the counts in grey matter and white matter in both disease groups (figure 3.2F and figure 3.3F). For the sALS cases, there was a significant correlation between neuronal and glial inclusions in both the white and grey matter (figure 3.3D,E). In contrast, neuronal inclusion pathology was not correlated with glial pathology in the motor cortex of C9ALS cases (figure 3.2D,E).

The anterior frontal cortex, as with the spinal cord and motor cortex, showed positive correlations for the white and grey matter glial inclusion pathology in both C9ALS and sALS cases (figure 3.2I and figure 3.3I). The neuronal pathology in the frontal cortex of both C9ALS and sALS groups was positively correlated with glial pathology in the grey matter (figure 3.2G and figure 3.3G) but not in the white matter (figure 3.2H and figure 3.3H).

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Area Tested variables z p Tau

C9orf72 ALS

SC neuronal vs. SC glial GM* 1.985 0.04714 0.369761 SC neuronal vs. SC glial WM** 2.8277 0.004689 0.5480769 SC glial GM vs. SC glial WM** 2.9729 0.00295 0.5741693 MCx neuronal vs. MCx glial GM 0.5457 0.5853 0.1057741 MCx neuronal vs. MCx glial WM 0.9449 0.3447 0.184466 MCx glial GM vs. MCx glial WM* 2.2324 0.02559 0.4327123 FCx neuronal vs. FCx glial GM** 2.8873 0.003886 0.5378341 FCx neuronal vs. FCx glial WM 1.5453 0.1223 0.293147 FCx glial GM vs. FCx glial WM** 2.9938 0.002755 0.5642881 MCx neuronal vs. SC glial WM -0.2745 0.7837 -0.05555556 MCx glial GM vs. SC glial WM -0.4386 0.6609 -0.08839914 MCx glial WM vs. SC glial WM -0.11 0.9124 -0.02234672

Sporadic ALS

SC neuronal vs. SC glial GM 1.8917 0.05854 0.2111265 SC neuronal vs. SC glial WM 1.8934 0.05831 0.214516 SC glial GM vs. SC glial WM*** 5.9384 2.878 × 10^-9 0.6601866 MCx neuronal vs. MCx glial GM*** 4.0755 4.591 × 10^-5 0.4491098 MCx neuronal vs. MCx glial WM*** 3.3018 9.605 × 10^-4 0.3677013 MCx glial GM vs. MCx glial WM** 2.6521 0.007999 0.2733926 FCx neuronal vs. FCx glial GM*** 3.3799 7.252 × 10^-4 0.3792862 FCx neuronal vs. FCx glial WM 1.1753 0.2399 0.1342031 FCx glial GM vs. FCx glial WM*** 3.9674 7.265 × 10^-5 0.414105 MCx neuronal vs. SC glial WM 1.7054 0.08812 0.225486 MCx glial GM vs. SC glial WM* 2.1912 0.02843 0.2724669 MCx glial WM vs. SC glial WM 0.5139 0.6073 0.06439714 Table 3.4: Correlations between p62-positive inclusion pathology of different areas. SC=spinal cord, MCx=motor cortex, FCx=frontal cortex, GM=layer V of the grey matter (for MCx and FCx) or ventral horns (for SC), WM=white matter under the cortex (for MCx and FCx) or corticospinal motor tracts (for SC). p, tau statistic, and z values calculated using the Kendall tau rank correlation coefficient. *p<0.05, **p<0.01, ***p<0.001.

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A positive correlation exists between the p62-positive pathology of the ventral horn motor neurones and that of the surrounding glia in the grey matter, suggesting that a common factor is affecting all cell types equally. A similar result was seen in the prefrontal cortex, but not in the motor cortex.

This could mean that the pathology in the precentral gyrus of C9ALS cases has a different origin for neuronal and glial cells.

Neuronal vs.

white matter glia

No correlations could be found between these in the examined cortical areas, meaning that neuronal and subcortical glial (myelinating the proximal part of their axons) pathologies occur independently of each other, maybe because of different origins. However, a positive correlation was found between ventral horn neurone pathology and that of the glia in the descending dorsolateral motor tracts of the spinal cord. This suggests that the pathology levels of the glia supporting the axons of the upper motor neurones could directly affect lower motor neurone degeneration or vice versa.

White matter vs.

grey matter glia

The levels of pathology between glia in the grey and the white matter correlated in all the studied areas, probably meaning that there is a common factor that causes all glia in a given area to degenerate.

Cortical vs.

spinal cord

No relationship could be seen between the pathology levels seen in the precentral gyrus and those of the supporting glia in the spinal cord motor tracts, meaning that they might happen independently of each other.

Sporadic ALS

Neuronal vs.

grey matter glia

Sporadic cases show positive correlations between cortical neuronal pathology and that of the surrounding glia. Most of these cases have no p62-related pathology in the cerebral cortex, but where it can be found, it affects neurones and glia at the same level. However, the ventral horns show no correlation between motor neurone and glial pathologies unlike C9ALS cases, where glial pathology is more common.

Neuronal vs.

white matter glia

Amongst the studied areas, only the precentral gyrus of sALS cases shows a correlation between pyramidal neurone pathology and that of the proximal glia in the white matter below. Added to the above, these results might indicate that the motor pathways of the few sALS cases with high pathology in this area share a common affection between neurones and glia.

White matter vs.

grey matter glia

As seen in the C9ALS cases, the levels of glial pathology correlate within each of the three studied areas. It seems that what affects glial degeneration in a particular area does not distinguish grey matter from white matter glia and all are affected in the cases where pathology is present.

Cortical vs.

spinal cord

The only correlation that reached significance was that between motor cortical glia and the supporting glia in the dorsolateral motor tracts of the spinal cord. As the latter did not correlate with the white matter glia under the motor cortex too, it is hard to find a logical reason for this correlation, which might be a false positive.

Table 3.5: Interpretation of the correlation data for p62-positive pathology.

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144 Figure 3.2: Pathological correlations in C9ALS. Significant correlations were observed in the spinal cord between glial p62 inclusion pathology in the ventral horns and the corticospinal tracts, and that on ventral horn neurones (A, B). The pathology found between those glial cells themselves also significantly correlated (C). Only glial cell pathology significantly correlates in the motor cortex, between that on the grey matter and that on the underlying white matter (F). The same is seen in the frontal cortex (I), where neuronal inclusion pathology also correlates with grey matter glial pathology. No correlation is seen between the pathology in the motor cortex and that observed in the corticospinal tracts.

*/**p < 0.05/0.01. p values calculated using the Kendall tau rank correlation coefficient. The shadowed area shows the 95% confidence interval.

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146 Figure 3.3: Pathological correlations in sALS. Glial p62 inclusion pathology in the ventral horns of the spinal cord correlates with that found in the corticospinal tracts (C), but none of the pathology on those areas correlates with ventral horn neuronal pathology. Conversely, motor cortex neuronal pathology is correlated to glial pathology in the neighbouring grey matter (D) and in the subcortical white matter (E). However, glial pathology in the motor cortex and the underlying white matter do not show a correlation (F). In the frontal cortex, neuronal pathology is correlated to grey matter glial pathology (G), but not to that in the white matter (H). Glial pathology in these two locations correlates significantly (I). Only a weak significant correlation was found when comparing grey matter glial pathology in the motor cortex with corticospinal pathology (K), but no relationship in pathology between these areas was generally found (J, L).

*/**/***p < 0.05/0.01/0.001. p values calculated using the Kendall tau rank correlation coefficient. The shadowed area shows the 95% confidence interval.

147 Thus, overall, for the spinal cord, motor cortex and frontal cortex glial pathology, there was a correlation in both groups between the grey and white matter within each region. The relationship between neuronal and glial pathology within these regions is more complex: for the spinal cord, a relationship between neuronal and glial pathology was seen only in the C9ALS but not sALS cases in spite of greater statistical power in the latter group. However, for the anterior frontal and motor cortices, the sALS cases showed positive correlations between neuronal and grey matter glial pathology. This was not seen in C9ALS cases potentially due to less statistical power or due to more than one type of p62-positive protein pathology caused by expanded C9orf72 mRNA.

We also investigated the relationships between pathology levels in different CNS regions (figure 3.2J-L and figure 3.3J-L). Firstly, we examined whether there was a relationship between inclusion pathology present in motor cortex neurones and the glia that support their axons in the spinal cord dorsolateral spinal tracts.

Secondly, we investigated whether there was a relationship between the inclusion counts in glia of those dorsolateral spinal tracts and the glial pathology in the motor cortex grey matter or white matter. There was very little evidence for any of these relationships save for a correlation between the number of layer V glial inclusions in motor cortex and corticospinal glial inclusions in the spinal cord of sALS but not C9ALS cases (p=0.02843, tau=0.2724, z=2.1912). In general, glial pathology levels showed greater correlations within CNS regions than between them, indicating that the p62-related pathology burden in the 2 different motor areas is independent of each other.

148 3.3.5 Relationship of p62-positive inclusion pathology to disease severity

We wished to assess the possibility that the different levels of glial p62-positive inclusion pathology found above were related to the progression of the illness or to the different age of the patients at death. To assess this, correlation tests were performed between the levels of pathology found in each area, and the duration of the disease and the age at death.

No correlations were found between glial p62-positive inclusions and the duration of the disease (p ≥ 0.08912, tau=-0.145, z=-1.7001) or the age at death (p ≥ 0.2593, tau=-0.1158, z=-1.1279) in any of the studied areas in either disease group.