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. 2023;12(3):267-281.
doi: 10.3233/JHD-230588.

Age-Dependent Increase in Tau Phosphorylation at Serine 396 in Huntington's Disease Prefrontal Cortex

Tiziana Petrozziello  1 , Sommer S Huntress  1 , Ayleen L Castillo-Torres  1 , James P Quinn  2 , Theresa R Connors  2 , Corinne A Auger  2 , Alexandra N Mills  1 , Spencer E Kim  1 , Sophia Liu  2 , Farah Mahmood  2 , Adel Boudi  2 , Muzhou Wu  3 , Ellen Sapp  2 , Pia Kivisäkk  2 , Shekar R Sunderesh  2 , Mahmoud A Pouladi  4 , Steven E Arnold  2 , Bradley T Hyman  2 , H Diana Rosas  2 , Marian DiFiglia  2 , Ricardo Mouro Pinto  2   3   5 , Kimberly Kegel-Gleason  2 , Ghazaleh Sadri-Vakili  1
Affiliations

Age-Dependent Increase in Tau Phosphorylation at Serine 396 in Huntington's Disease Prefrontal Cortex

Tiziana Petrozziello et al. J Huntingtons Dis. 2023.

Abstract

Background: To date, it is still controversial whether tau phosphorylation plays a role in Huntington's disease (HD), as previous studies demonstrated either no alterations or increases in phosphorylated tau (pTau) in HD postmortem brain and mouse models.

Objective: The goal of this study was to determine whether total tau and pTau levels are altered in HD.

Methods: Immunohistochemistry, cellular fractionations, and western blots were used to measure total tau and pTau levels in a large cohort of HD and control postmortem prefrontal cortex (PFC). Furthermore, western blots were performed to assess tau, and pTau levels in HD and control isogenic embryonic stem cell (ESC)-derived cortical neurons and neuronal stem cells (NSCs). Similarly, western blots were used to assess tau and pTau levels in HttQ111 and transgenic R6/2 mice. Lastly, total tau levels were assessed in HD and healthy control plasma using Quanterix Simoa assay.

Results: Our results revealed that, while there was no difference in total tau or pTau levels in HD PFC compared to controls, the levels of tau phosphorylated at S396 were increased in PFC samples from HD patients 60 years or older at time of death. Additionally, tau and pTau levels were not changed in HD ESC-derived cortical neurons and NSCs. Similarly, total tau or pTau levels were not altered in HttQ111 and transgenic R6/2 mice compared to wild-type littermates. Lastly, tau levels were not changed in plasma from a small cohort of HD patients compared to controls.

Conclusions: Together these findings demonstrate that pTau-S396 levels increase significantly with age in HD PFC.

Keywords: ESC-derived neurons; Huntington’s disease; fractionations; mouse models; neuronal stem cells; phosphorylated tau; plasma; postmortem brain; total tau.

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Conflict of interest statement

P.K. is named as co-inventor on a U.S. patent application related to neurological biomarker assays that is jointly held by Massachusetts General Hospital and Meso Scale Diagnostics. R.M.P. has received sponsored research funding from Pfizer Inc. None of this had any influence over this manuscript.

Figures

Figure 1.
Figure 1.. Total tau and pTau-S396 levels were not altered in HD PFC.
(A) Representative western blot images of tau, pTau-S396, pTau-S404, pTau-T181 and GAPDH in whole cell homogenates derived from control and HD PFC revealing no alterations in tau and pTau levels. (B) There were no alterations in tau in HD PFC (n = 20) compared to controls (n = 14) (Mann-Whitney U test = 95, p = 0.2069). (C) pTau-S396 levels were not altered in HD PFC (n = 20) compared to controls (n = 14) (Mann-Whitney U test = 87.50, p = 0.0997). (D) There were no alterations in pTau-S404 between HD (n = 19) and control PFC (n = 14) (Mann-Whitney U test = 82, p = 0.1697). (E) pTau-T181 levels were not altered in HD PFC (n = 19) compared to controls (n = 11) (Mann-Whitney U test = 71.50, p = 0.3879).
Figure 2.
Figure 2.. pTau-S396 levels were increased in elderly HD PFC.
(A) pTau-S396 levels did not change in PFC derived from younger HD patients (< 60 y; n = 9) compared to age-matched controls (n = 3) (Mann-Whitney U test = 12.50, p = 0.8955). (B) There was a significant increase in pTau-S396 levels in PFC derived from older HD patients (> 60 y; n = 11) compared to age-matched controls (n = 10) (Mann-Whitney U test = 17, p = 0.0110). (C) No alterations in pTau-S404 levels were found in young HD PFC (n = 7) compared to controls (n = 3) (Mann-Whitney U test = 6, p = 0.3833). (D) pTau-S404 levels were not altered in older HD PFC (n = 12) compared to age-matched controls (n = 11) (Mann-Whitney U test = 38, p = 0.2512). (E) There was no change in pTau-T181 levels between HD (n = 7) and control PFC (n = 2) derived from younger patients (Mann-Whitney U test = 6, p = 0.8889). (F) pTau-T181 levels were not altered in older HD PFC (n = 12) compared to controls (n = 9) (Mann-Whitney U test = 44.50, p = 0.7250). * p < 0.05.
Figure 3.
Figure 3.. pTau-S396 levels were not altered in gray and white matter in HD PFC.
(A) Representative IHC images of pTau-S396 staining in grey matter from AD entorhinal cortex, control and HD PFC. Extensive neuropil threads and NFTs were observed in AD cortex, while no threads or NFTs were evident in both control and HD PFC. (B) There were no alterations in pTau-S396 levels in the grey matter from HD PFC (n = 24) compared to controls (n = 18) (Mann-Whitney U test = 175, p = 0.3069). (C) Representative IHC images of pTau-S396 staining in the white matter from AD entorhinal cortex, control and HD PFC. Neuropil threads and NFTs were not observed in control and HD PFC. (D) No significant changes were observed in pTau-S396 levels in HD PFC white matter (n = 24) compared to control PFC (n = 18) (Mann-Whitney U test = 210.5, p = 0.8950). Scale bar: 100 μm; insert scale bar: 5 mm.
Figure 4.
Figure 4.. Soluble and insoluble species of total tau and pTau-S396 were not altered in HD PFC.
(A) Representative western blot images of tau, pTau-S396 and amido black in total extract fractions derived from control and HD PFC revealing no alterations in tau and pTau-S396. (B) No differences were observed in tau levels in the total extract fraction from HD PFC (n = 8) compared to controls (n = 9) (Mann-Whitney U test = 23, p = 0.2359). (C) There were no changes in pTau-S396 levels in total extract fraction from HD (n = 8) and control PFC (n = 9) (Mann-Whitney U test = 33, p = 0.8148). (D) Representative western blot images of tau, pTau-S396 and amido black in total soluble fractions from control and HD PFC demonstrating no changes in tau and pTau-S396. (E) Tau levels were not changed in HD total soluble fraction (n = 8) compared to controls (n = 9) (Mann-Whitney U test = 24, p = 0.4698). (F) No significant alterations were observed in pTau-S396 levels in HD total soluble fraction (n = 8) compared to control soluble fraction (n = 9) (Mann-Whitney U test = 29, p = 0.5414). (G) Representative western blot images of tau, pTau-S396 and amido black in Triton X-100 soluble fractions derived from control and HD PFC revealing no alterations in tau and pTau-S396. (H) There were no changes in tau levels in the Triton X-100 soluble fraction from HD PFC (n = 8) and controls (n = 9) (Mann-Whitney U test = 23, p = 0.2359). (I) No alterations were found in pTau-S396 levels in Triton X-100 soluble fraction between HD PFC (n = 8) and controls (n = 9) (Mann-Whitney U test = 23, p = 0.6126). (J) Representative western blot images of tau, pTau-S396 and amido black in Sarkosyl soluble fractions from control and HD PFC demonstrating no differences in tau and pTau-S396. (K) Tau levels were not altered in the Sarkosyl soluble fractions in HD (n = 8) compared to control PFC (n = 9) (Mann-Whitney U test = 12, p = 0.0721). (L) There were no changes in pTau-S396 levels in the Sarkosyl soluble fraction in HD (n = 8) compared to control PFC (n = 9) (Mann-Whitney U test = 31, p = 0.6730). (M) Representative western blot images of tau, pTau-S396 and amido black in Sarkosyl insoluble fractions from control and HD PFC revealing no alterations in tau and pTau-S396. (N) Tau levels were not changed in the Sarkosyl insoluble fractions derived from HD (n = 8) and control PFC (n = 9) (Mann-Whitney U test = 27, p = 0.6454). (O) There was no change in pTau-S396 levels in the Sarkosyl insoluble fraction between HD PFC (n = 8) and controls (n = 9) (Mann-Whitney U test = 23, p = 0.2359).
Figure 5.
Figure 5.. Synaptic total tau and pTau-S396 levels were not altered in HD PFC.
(A) Representative western blot images of tau, pTau-S396, PSD-95, and GAPDH in total extracts, cytosolic fractions and SNs derived from control and HD PFC. There were no alterations in tau and pTau-S396 levels between HD and control PFC. PSD-95 immunosignal was detected in total extracts and SNs demonstrating purity of the fractionation. (B) Two-way ANOVA revealed no significant effect of cellular fractions ([F(2,58) = 1.812], p = 0.1724), genotype [F(1,58) = 0.2429], p = 0.6240) and cellular fraction × genotype interaction ([F(2,58) = 0.2701], p = 0.7643) on tau levels. Tukey’s test confirmed no change in tau levels in the three fractions derived from HD (n = 14) and control PFC (n = 5) (p > 0.05). (C) Two-way ANOVA demonstrated a significant effect of cellular fractions ([F(2,45) = 4.133], p = 0.0255), while there were no significant effects of genotype [F(2,45) = 0.1766], p = 0.6763) and cellular fraction × genotype interaction ([F(2,45) = 0.1895], p = 0.8280) on pTau-S396 levels. Tukey’s test revealed no alterations between the three fractions in HD (n = 15) and control samples (n = 5) (p > 0.05).
Figure 6.
Figure 6.. There were no alterations in total tau and pTau-S396 levels in HD ESC-derived cortical neurons and NSCs.
(A) Representative western blot images of tau, pTau-S396, and GAPDH in ESC-derived cortical neurons from HD and controls revealing no alterations in tau and pTau-S396 levels. Cell lines were derived from a single person living with HD and a single healthy control individual and every experiment was performed in triplicates. (B) There were no significant differences in total tau levels in HD ESC-derived neurons compared to the isogenic control cell line (Mann-Whitney U test = 2, p = 0.1143). (C) No differences in pTau-S396 levels were observed in ESC-derived cortical neurons between HD and control cell lines (Mann-Whitney U test = 6, p = 0.6857). (D) Representative western blot images of tau, pTau-S396, and GAPDH in NSCs derived from HD and control patients demonstrating no differences in tau and pTau-S396 levels. (E) There were no alterations in total tau levels in HD ESC-derived NSCs compared to isogenic control cells (Mann-Whitney U test = 4, p >0.9999). (F) pTau-S396 levels did not change in HD NSCs compared to isogenic control cells (Mann-Whitney U test = 3, p = 0.7000).
Figure 7.
Figure 7.. There were no differences in total tau and pTau-S396 levels in two different HD mouse models.
(A) Representative western blot images of tau, pTau-S396 and GAPDH in wild-type (Wt) and HttQ111 cortex revealing no alterations in tau and pTau-S396 levels. (B) No changes in tau levels were reported in HttQ111 (n = 8) compared to Wt cortex (n = 8) (Mann-Whitney U test = 15, p = 0.0830) (C) There were no alterations in pTau-S396 levels in HttQ111 (n = 8) compared to Wt cortex (n = 8) (Mann-Whitney U test = 18, p = 0.1521). (D) Representative western blot images of tau, pTau-S396 and GAPDH in R6/2 transgenic mice and Wt littermates demonstrating no differences in tau and pTau-S396 levels. (E) No alterations in total tau levels were observed in transgenic R6/2 (Tg) (n = 8) compared to Wt cortex (n = 8) (Mann-Whitney U test = 28, p = 0.7209). (F) pTau-S396 levels were not altered in Tg R6/2 (n = 8) compared to Wt cortex (n = 8) (Mann-Whitney U test = 23, p = 0.3671).
Figure 8.
Figure 8.. Tau levels were not altered in HD plasma.
There were no alterations in plasma tau levels between HD (n = 9) and healthy control patients (n = 10) (Mann-Whitney U test = 38, p = 0.6038). One sample had a tau level under the LLOQ and was not included in the figure.
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