Threonine175, a novel pathological phosphorylation site on tau protein linked to multiple tauopathies

Microtubule associated protein tau (tau) deposition is associated with a spectrum of neurodegenerative diseases collectively termed tauopathies. We have previously shown that amyotrophic lateral sclerosis (ALS) with cognitive impairment (ALSci) is associated with tau phosphorylation at Thr175 and that this leads to activation of GSK3β which then induces phosphorylation at tau Thr231. This latter step leads to dissociation of tau from microtubules and pathological tau fibril formation. To determine the extent to which this pathway is unique to ALS, we have investigated the expression of pThr175 tau and pThr231 tau across a range of frontotemporal degenerations. Representative sections from the superior frontal cortex, anterior cingulate cortex (ACC), amygdala, hippocampal formation, basal ganglia, and substantia nigra were selected from neuropathologically confirmed cases of Alzheimer’s disease (AD; n = 3), vascular dementia (n = 2), frontotemporal lobar degeneration (FTLD; n = 4), ALS (n = 5), ALSci (n = 6), Parkinson’s disease (PD; n = 5), corticobasal degeneration (CBD; n = 2), diffuse Lewy body dementia (DLBD; n = 2), mixed DLBD (n = 3), multisystem atrophy (MSA; n = 6) and Pick’s disease (n = 1) and three neuropathologically-normal control groups aged 50–60 (n = 6), 60–70 (n = 6) and 70–80 (n = 8). Sections were examined using a panel of phospho-tau antibodies (pSer208,210, pThr217, pThr175, pThr231, pSer202 and T22 (oligomeric tau)). Across diseases, phospho-tau load was most prominent in layers II/III of the entorhinal cortex, amygdala and hippocampus. This is in contrast to the preferential deposition of phospho-tau in the ACC and frontal cortex in ALSci. Controls showed pThr175 tau expression only in the 7th decade of life and only in the presence of tau pathology and tau oligomers. With the exception of DLBD, we observed pThr175 co-localizing with pThr231 in the same cell populations as T22 positivity. This suggests that this pathway may be a common mechanism of toxicity across the tauopathies. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0406-4) contains supplementary material, which is available to authorized users.


Introduction
Microtubule associated protein tau (tau) is a cytoskeletal stabilizing protein involved in microtubule maintenance, fast axonal transport, and other physiological functions in neurons. Tau protein deposition is a characteristic of many neurodegenerative diseases that are collectively referred to as tauopathies. It has been shown that pathological species of tau protein are abnormally phosphorylated at multiple residues [1] and that this is linked to a decrease in tau's ability to bind to and stabilize microtubules [2,3] with accompanying cytotoxicity [4]. While the isoform composition of insoluble tau deposits and the structural formation of the protein aggregates differs amongst the tauopathies, there are several phosphorylation sites that are thought to be universally important in the induction of a tauopathy.
One potentially important phosphorylation site that has gone relatively unstudied is threonine 175 (Thr 175 ). First identified in Alzheimer's disease as a unique phosphoepitope [5], it was then determined that this site could be phosphorylated by multiple kinases linked to tau protein pathology, including GSK3β, JNK, ERK2, and p38 [6]. pThr 175 tau was then identified in amyotrophic lateral sclerosis with cognitive impairment (ALSci) [7] and characterized in further detail in the context of this disease [8][9][10][11][12]. Importantly, pThr 175 tau has been shown to induce tau fibril formation and cell death in vitro [13]. Unlike other widely accepted pathological phosphorylation sites on tau, such as pThr 231 and pSer 202 , pThr 175 has not been observed in the fetal brain where tau protein is hyperphosphorylated [14][15][16], suggesting that this site may be uniquely associated with pathological processes. pThr 175 tau has been shown to induce GSK3β activation in cell culture and may therefore act as a destabilizing event resulting in enhanced phosphorylation of tau at other residues, resulting in dissociation from microtubules and neuronal toxicity [17]. In order to understand the extent to which this pathway of pThr 175 mediated tau aggregate formation underlies a broad range of tauopathies, we have used a panel of phospho-specific antibodies to characterize tau protein pathology with specific interest in the expression of pThr 175 tau across a broad range of tauopathies.
To assess the extent of pThr 175 tau, pThr 231 tau and tau oligomer pathological inclusions as a function of ageing, three groups of controls were studied, encompassing the 6 th (n = 6), 7 th (n = 6), and 8 th (n = 8) decades of life (Table 1). Hippocampal sections from each group were stained for pThr 175 tau, pThr 231 tau and oligomeric tau (T22). These cases have been previously characterized in a study examining age-dependant tau deposition in the frontal and entorhinal cortices and were shown to be free of neurodegenerative disease [22].
Five to six micrometer paraffin-embedded sections from the superior frontal gyrus, anterior cingulate (ACC), hippocampus, entorhinal cortex, dentate gyrus, amygdala, basal ganglia and substantia nigra were used for all immunohistochemical analyses.
Antigen retrieval was conducted as necessary (Table 2). Endogenous peroxidase was quenched with 3% hydrogen peroxide (BDH Chemicals, VWR, On, Canada). Primary antibody incubation was performed at 4°C overnight in blocking buffer (5% BSA, 0.3% Triton-X 100 in 1 X PBS). After washing, secondary antibody (1:200 biotinylated IgG) incubation was performed for 1 h at room temperature in blocking buffer. Antigen:antibody complex was visualized with either horseradish peroxidase or alkaline phosphatase according to the manufacturer's instructions (Vectastain ABC kit, Vector Laboratories CA, USA), followed by substrate development with either DAB plus NiCl 2 or AP substrate kit III (Vector Laboratories). Counterstaining was performed using haematoxylin or nuclear fast red. The extent of pathology was described topographically and semiquantitatively as previously reported [12]. Representative images were captured with a 20x lens under light microscopy (Olympus BX45) and subsequently used for semi- quantitative analysis. The semi-quantitative scale was manually applied for each type of pathology by an evaluator blinded to the underlying diagnosis (WY) (neuronal, neuritic, or glial) separately as follows: '-' = none; '±' = less than 5 inclusions; '+' = less than 10 inclusions; '++' = more than 20 inclusions with scattered distribution; '+++' = more than 20 inclusions but with locally dense distribution; '++++' = more than 20 inclusions with a diffuse distribution. Additionally, the case positive ratio was defined for each antibody used and brain region investigated as the number of cases showing any pathology (± or more) compared to the total number of cases stained.

Oligomeric tau and pThr 231 staining
Rabbit anti T22 (EMD Millipore CA, USA) and rabbit anti tau pThr 231 (Thermo Fischer) were used to probe tau inclusions for the presence of oligomeric tau (T22) and for phosphorylation at Thr 231 . Tau oligomeric species are currently hypothesized to be more toxic to neurons than the fibrillar inclusions themselves [23], and pThr 231 is thought to be a key site in the regulation of tau protein folding and ability to interact with microtubules [3,24]. Double labeling was performed on hippocampus from one case each from AD, ALSci, FTD, MSA, DLDB, and mDLDB. Tau protein was probed for pThr 175

Results
Tau antibody staining Neuronal tau ALS Consistent with our earlier reports, we observed tau pathology in multiple brain regions in ALS, although to a lesser degree than either ALSci or the remaining tauopathies. Neuronal tau inclusions were most consistently observed in the entorhinal cortex, hippocampus, and amygdala. All antibodies were immunoreactive with neuronal tau inclusions in multiple brain regions (Table 3). Frontal and anterior cingulate pathology was limited in both load and case-positive incidence. In all regions studied, inclusions took the form of punctate cytosolic inclusions or tangles (Fig. 1). Deposition was mainly restricted to the superficial cortical layers in the entorhinal cortex but restricted to deeper layers in the ACC and superior frontal cortex when present.
ALSci Tau pathology ( Fig. 1, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3) in the form of tangles, skein-like inclusions, and punctate staining was observed to a greater extent in ALSci than ALS, especially in the ACC and superior frontal cortex. The load of pathology was increased in amount and distribution and the case positive ratio was higher than ALS in all brain regions studied ( Table 4). As observed in ALS, pathological tau neuronal inclusions were observed predominantly in the superficial layers of the entorhinal cortex and within deeper cortical layers in the ACC and superior frontal cortex. However superficial layer involvement was noted in both the ACC and superior frontal cortex in ALSci, indicating a greater distribution across cortical layers in ACC and frontal cortex, further differentiating ALSci from ALS. Of note, Thr 175 tau and PHF tau identified pathology to different extents in different brain regions. Notably, pThr 175 tau and pThr 217 tau identified a higher case positive ratio than PHF in the superior frontal cortex.
Tauopathies Within the tauopathies and consistent with the literature, we observed tau neuronal pathology across multiple regions (Table 5). pThr 175 tau was present in all disease states where tau pathology was prominent. In Alzheimer's disease (AD), all tau antibodies showed robust neuronal pathology as neurofibrillary tangles and punctate cytoplasmic inclusions in all 1) Boil in 10 mM sodium citrate, 0.05% Tween 20 pH 6.0 for 2 min 2) Pressure cooker (2100 Retriever; Aptum Biologics, UK) 10 mM sodium citrate, 0.05% Tween 20 pH 6.0 for 15 min brain regions studied. Across all cortical regions studied, neuronal pathology was present across all cortical layers but was most prominent in deeper layers (IV-VI). Amongst the tauopathies, the most prominent pThr 175 tau immunostaining was observed in AD. This included more prominent expression of pThr 175 tau than observed in ALSci. As in AD, VD exhibited tau protein deposition as tangles and punctate cytoplasmic inclusions in all brain regions studied. This followed the same trend as AD with pathology being most prominent in deep cortical layers. In CBD, balloon neurons were observed and tau pathology was prominent in all brain regions as punctate inclusions and neurofibrillary tangles. Notably, PHF tau staining was more intense than pThr 175 tau in all regions, both in case positive ratio and in semiquantitative pathological load. In both DLBD and mDLBD, a similar degree of tau pathology was observed in the form of cytoplasmic punctate deposition and neurofibrillary tangles. Pathology within the dentate gyrus, basal ganglia and substantia nigra was present to a much greater extent in mDLBD than DLBD. In FTLD, tau pathology was observed as punctate inclusions and tangles in all brain regions investigated. In general, pThr 175 tau was less prominent than PHF tau, except in the frontal and cingulate cortex where it was more prominent on a case positive basis and the pathological load observed. In MSA, tau pathology in the form of tangles and punctate inclusions was present in all brain regions studied although frontal and ACC pathology was sparse. In Parkinson's disease, tau pathology was observed in all brain regions except the substantia nigra. Pathological tau expression was equivalent across all antibodies. In Pick's disease, all brain regions investigated exhibited tau pathology in the form of tangles, punctate inclusions and Pick bodies. Notably, PHF tau pathology was greater than the other antibodies including pThr 175 tau.

Neuritic tau
ALS No neuritic plaques were observed in ALS. Neuritic pathology in the form of dystrophic neurites was observed to a limited extent in all brain regions studied and with a pattern of distribution mimicking that described above for neuronal pathology. Basal ganglia neuritic pathology was observed to a larger extent in the putamen than the globus pallidus by all tau antibodies but pThr 175 tau. Neuritic tau pathology within the substantia nigra was immunoreactive against all antibodies employed in the analysis.
ALSci Neuritic tau pathology was observed predominantly as dystrophic neurites assuming a short curvilinear morphology. This was consistently observed in both cortical and subcortical tissues. Contrary to the superficial localization of frontal and ACC neuronal pathology, frontal neuritic pathology was observed mainly in deep layers as short curved neurites. Entorhinal neuritic pathology was observed mainly in the superficial layers in proximity to tau inclusion bearing neurons. Neuritic plaques were observed in the entorhinal cortex by all antibodies with the exception of the pThr 175 tau antibody. Neuritic plaques within the amygdala were observed by PHF tau antibody labeling only. Neuritic plaques and tau positive neurites were observed in the hippocampus and  were immunoreactive to all antibodies. Coiled bodies were observed throughout the basal ganglia.
Tauopathies Neuritic pathology was prominent across all the tauopathies and typically mirrored the presence of tau immunoreactive neuronal pathology. Neuritic plaques were observed in AD as small atypical plaques and typical plaques in deeper layers (IV/V) more frequently than in superficial layers (II/III). No antibody identified neuritic plaques in basal ganglia or substantia nigra. Like neuronal tau, all cortical neuritic pathology was most prominent in deeper layers (IV-VI).
All antibodies recognized neuritic pathology, most commonly in deeper cortical layers near tau positive neurons as short and curved, or long, straight neurites. Neuritic pathology was also observed to a lesser extent in white matter in frontal, ACC, and entorhinal cortices. Similar to AD, neuritic pathology in VD was present as tau positive neurites and neuritic plaques in all brain regions studied except the substantia nigra. Also similar to AD, neuritic pathology followed a tendancy to be most pronounced in regions of prominent neuronal tau pathology and in particular in the deeper cortical layers. In CBD, neuritic plaques were only observed in the ACC and substantia nigra, and then, only using the PHF tau antibody. Neuritic pathology, as dystrophic neurites, was present in all brain regions investigated and mirrored the distribution of neuronal pathology. This was most evident in the entorhinal cortices where dystrophic neurites were most evident in superficial cortical layers. In contrast, dystrophic neurites were most prominent in deep subcortical regions of the superior frontal cortex and ACC. In DLBD, no frontal, ACC, dentate gyrus or basal ganglia neuritic pathology was observed. All other regions studied were positive for neuritic tau pathology, but no plaques were observed. Entorhinal neuritic pathology was most prominent in DLDB in layers II/III. In mDLBD, neuritic plaques were observed in all regions except for the substantia nigra. All regions studied exhibited neuritic pathology. In both FTLD and MSA, neuritic plaque pathology was not frequent and usually observed by PHF tau only. pThr 175 tau neuritic pathology was not as prominent as that observed using other tau antibodies. However, neuritic tau pathology was observed in all brain regions studied as with neuronal pathology. In PD, neuritic plaques were not observed, but tau positive neurites were observed in a similar distribution to neuronal pathology in all brain regions studied. In Pick's disease, neuritic pathology was identified mainly by PHF tau in all but the dentate gyrus.

Glial tau
ALS Glial tau pathology was observed in all brain regions except the dentate gyrus, and substantia nigra ( Table 3). Where present, glial pathology presented as glial fibrillary tangles and astrocytic plaques as previously described [12]. The distribution was sparse and followed that of neuronal and neuritic tau as described above and was similar in case positive ratio, although higher in semiquantitative load than neuronal and neuritic pathology in a single case.
ALSci Glial pathology was present in ALSci ( Fig. 1, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3) to a similar degree as in ALS, but was more frequent in terms of regional distribution, case positive rate, and pathological load (Table 4). Frontal and ACC glial pathology was increased in both case positive incidence and pathological load.
Tauopathies In the tauopathies, the extent of glial pathology was highly dependant on the underlying disease (Table 5). In AD, glial pathology was rare, and when present was usually observed only in the amygdala and basal ganglia. No glial pathology was observed in VD. Consistent with the literature, CBD contained astrocytic plaques throughout the grey and white matter across multiple brain regions. Additional astrocytic staining and tau positive microglia were observed. Glial pathology in CBD was identified to a far greater extent by PHF tau than the pThr 175 tau antibody. In DLBD, minimal glial pathology was observed in frontal cortex and basal ganglia, and when present,  only as punctate astrocytic inclusions. In contrast to the limited glial pathology observed in DLBD, mDLBD showed much more frequent glial pathology across multiple brain regions, pathology that was less evident with the pThr 175 tau antibody. In FTLD, glial pathology was observed in all brain regions except the dentate gyrus and substantia nigra. Notable in this disease, pThr 175 tau identified glial pathology to a greater extent than PHF tau by both case positive incidence and increased load. In MSA, glial pathology was largely absent, being present only in the entorhinal cortex, amygdala and basal ganglia by multiple antibodies. Interestingly, the basal ganglia contained astrocytic inclusions in the putamen identified by all antibodies whereas in the globus pallidus they were only identified by pThr 175 tau. In Parkinson's disease, glial pathology was present in all regions except the dentate gyrus and substantia nigra. Notably, the PHF tau antibody identified astrocytic plaques in the frontal cortex while tufted astrocytes were observed in the amygdala. In Pick's disease, glial pathology was observed in multiple brain regions using the PHF antibody mainly, although in the entorhinal cortex pThr 175 tau was also positive for glial pathology.
pThr 231 tau and T22 staining We examined the presence of oligomeric tau (recognized by the T22 antibody) and pathological tau phosphorylation at Thr 231 using sections from the hippocampus of a single case each of AD, ALSci, FTD, MSA, DLBD, and mDLB. Cases were selected on the basis of the pathology described earlier. In each case, tau neuronal inclusions were recognized by both antibodies (Figs. 2 and 3). Only DLBD showed notably reduced T22 pathology which, when present, was in the dystrophic neurites (Fig. 3). Glial pathology was not observed with either antibody, regardless of diagnosis. AD neuronal pathology was observed as fibrillar and punctate inclusions by pThr 231 tau antibody but only as fibrillar/tangles with T22. Neuritic pathology in the form of short and long torsional and dystrophic neurites was observed. pThr 231 tau, but not T22, recognized plaques consisting of dystrophic neurites. Similar to AD, ALSci neuronal pathology was observed by pThr 231 tau and T22 as fibrillar and punctate staining. However additional solitary cytoplasmic inclusions were observed in neurons with the T22 antibody. Neuritic pathology was observed using both the pThr 231 tau and T22 antibodies. Neuritic plaques were identified by both the pThr 231 tau and T22 antibodies.
FTLD pathology was distinct from AD and ALSci in that pThr 231 tau neuronal pathology was observed as a dense nuclear ring staining around abnormally folded nuclei, and solitary cytosolic inclusions on homogenously stained cytosol. While T22 neuronal cytoplasmic pathology was observed, neuritic pathology was observed as frequent short dystrophic neurites by both antibodies. No neuritic plaques were observed.
MSA pathology resembled AD and ALSci. Neuronal pathology was observed as tangles and punctate inclusions by both the pThr 231 tau and T22 antibodies. In addition, pThr 231 tau antibody diffuse cytoplasmic immunostaining was observed. Neuritic pathology was observed by both antibodies, although T22 immunoreactive neurites demonstrated a punctate staining pattern. DLBD tau pathology was observed only faintly by pThr 231 tau as punctate cytosolic deposition. While neuritic pathology was observed using the pThr 231 tau antibody, no pathology was observed by T22 other than a few sporadic neurites. Conversely, mDLBD pathology was observed by both pThr 231 tau and T22. pThr 231 tau pathology was observed as tangles and punctate staining accompanied by dystrophic neurites and other neuritic pathology. No neuritic plaques were observed. T22 pathology however was observed as punctate cytosolic staining and dystrophic neurites.
Having confirmed that both T22 and pThr 231 tau immunoreactive pathology was present, although as described to varying degrees, we next sought to confirm whether pThr 175 tau and pThr 231 tau co-localized using confocal imaging (Fig. 4). Colocalization within neuronal tau inclusions was observed between pThr 175 tau and pThr 231 tau in each disease state except for DLBD. Colocalization was also observed in neuritic plaques in AD. No pThr 175 tau immunoreactivity was observed in the absence of pThr 231 . Hippocampal pThr 175 , pThr 231 and oligomeric tau deposition as a function of aging Consistent with our previous report, we observed an increase in tau-immunoreactive pathology beginning in the 7 th decade of life [22]. In contrast, no immunoreactive inclusions were observed to either the pThr 175 tau or oligomeric tau (T22 immunoreactivity) in the 6 th decade (Fig. 5). pThr 231 tau immunoreactivity was observed in each of the 6 th , 7 th and 8 th decades within the hippocampus. In distinction to the pathological tau deposition observed in both ALSci and the tauopathies, neuronal Thr 231 tau immunoreactivity was diffuse and localized to otherwise healthy appearing neurons and axonal processes. In the 7 th decade, T22 immunoreactive neuronal cytoplasmic inclusions were observed minimally and when present were within the same regions in which we observed punctate pThr 175 immunoreactivity. All but one case demonstrated pThr 231 tau immunoreactivity, and importantly this case was negative for all three tau epitopes. In all cases, neuritic pathology was minimal or In the 8 th decade, we observed a marked increase in pThr 175 tau, pThr 231 tau and T22 immunoreactivity. In this decade, each antibody revealed tau immunoreactive punctate staining of neurons, neurofibrillary tangles, dystrophic neurites, and neuritic plaques. Across all three decades, T22 pathology was present in all cases and in regions where pThr 231 tau and pThr 175 tau was present, and was only positive in cases with prominent pThr 175 tau positive cells and pathology.

Discussion
In undertaking these studies, we were specifically interested in determining whether the pathogenic phospho-tau species recognized by antibodies against pThr 175 tau and pThr 231 tau as well as oligomeric tau (T22) were expressed across a broad range of tauopathies. We were also interested in determining whether these pathological tau species were colocalized in ALS and ALSci. It is known that the phosphorylation of tau protein at Thr 231 is of both physiological and pathological significance in mediating the dissociation of tau from microtubules [3,24,25]. Thr 231 is phosphorylated by activated GSK3β physiologically and in pathological states [25][26][27][28][29]. We have previously shown that pThr 175 tau induces GSK3β phosphorylation and that this in turn leads to Thr 231 tau phosphorylation resulting in tau fibril formation and cell death in vitro [17].
Although the number of cases studied here is limited, the intent was not to undertake a detailed topographic analysis of tau deposition across all tauopathies, but rather to determine whether the proposed pathway of pThr 175 tau mediated induction of pThr 231 tau formation with its attendant pathological tau fibril formation (as recognized by T22) was evident. It is noteworthy therefore that we observed that in each tauopathy studied, pThr 175 tau, pThr 231 tau and T22 immunoreactivity co-localized to the same inclusion-containing neuronal populations. In each case, neuronal pThr 175 tau colocalized with pThr 231 tau. This, paired with the prior identification of pThr 175 tau in AD brain tissue but not controls [30] and the lack of identified pThr 175 in fetal tau [15,16] suggests that pThr 175 is a key point in pathological tau metabolism, as it is not a physiologically utilized site involved in the regulation of tau function during development or microtubule reorganization. This suggests that the downstream events triggered by pThr 175 tau, including toxic monomer formation, are common to each of these diseases.
To further assess the pathogenicity of pThr 175 and pThr 231 , we investigated each epitope in the hippocampus of control cases across three decades of life where tau pathology has been shown to increase with age [22]. We observed no pThr 175 tau pathology in the 6 th decade with minimal immunoreactive neuronal inclusions in the 7 th decade. pThr 175 tau immunoreactivity was most Fig. 5 Age dependent tau pathology increases in the hippocampus of controls and is associated with pThr 175 tau pathology in the 8 th decade of life. Large images taken in the CA2 region of the hippocampus at 10× magnification, inset is same region at 40× magnification prominent in the 8 th decade. In each case in which we observed pThr 175 tau immunostaining, we also observed T22 immunoreactivity. Similarly, we never observed T22 immunoreactivity in the absence of either pThr 175 tau or pThr 231 tau immunoreactivity. In contrast, pThr 231 tau immunoreactivity was frequently observed in the absence of either pThr 175 tau or T22 staining in younger individuals and when present, was within healthy appearing neurons and axonal processes. pThr 175 and T22 did not show pathology in hippocampal regions spared from pThr 231 pathology, and T22 was only positive in cases showing prominent pThr 175 pathology.
Glial pathology was recognized to a greater degree by pThr 217 tau and PHF tau than by the pThr 175 tau antibody, suggesting that different pathological processes are at play in these cells. This is supported by the lack of identifiable glial pathology by pThr 231 tau and T22. This paired with the low frequency of pThr 175 tau glial pathology further strengthens the correlation between pThr 175 and pThr 231 in the induction of neuronal pathology and provides evidence that this pair of phosphorylation sites may be exerting specific neuronal toxicity in the disease process across multiple tauopathies.
Although limbic regions universally presented tau pathology, frontal and ACC tau pathology was present mainly in AD, VD, ALSci, FTLD, mDLBD and MSA. This paired with the deeper layer pathology in this region may indicate that tau pathology did not originate here but instead propagated from other regions. If tau originates in limbic structures, propagating along the Papez circuit, it is possible that it would arrive in ACC through thalamic projections to layer IV and V which could act as a hub for propagation to other brain regions such as frontal cortex through this well connected region. Regardless of the induction cause or place, tau protein toxicity is undeniable once initiated [23,31], and must be considered when attempting to understand the underlying biology of many neurodegenerative diseases. This hypothesis also implies that disease entities such as primary age-related tauopathies (PART) [32] may be in fact not age-related, but neuronal stress related, as increasing age would indicate longer time periods for stresses on neurons to become pathological through stochastic processes [33]. Therefore, tau protein deposition should not be considered a simple function of normal ageing, but ageing should be considered a risk factor for tauopathy among a plethora of neuronal stresses. Of note as well is the frontal involvement in ALSci, which can be concluded is not likely a result of PART, which spares the neocortex by definition [22,32]. We cannot conclude, however if the layer distribution of tau pathology resembles PART, as this was not described in the consensus report.

Conclusions
These findings implicate a toxic axis of phosphorylation events beginning with Thr 175 phosphorylation, dependent on further phosphorylation at Thr 231 , which appears to be neuron specific and which may be common to the tauopathies. The extent to which this process is reflective of not only the presence of a tauopathy, but the disease load as would be encapsulated in the "ABC" score will require much greater case numbers across each disease state. In this paper, we simply raise the critical question that this pathway of pathological tau phosphorylation may be a contributor to neuronal death in these diseases, and thus a point of intervention capable of slowing disease progression resulting from tau protein toxicity.