Evidence of mutant huntingtin and tau-related pathology within neuronal grafts in Huntington's disease cases

Abstract

A number of post-mortem studies conducted in transplanted Huntington's disease (HD) patients from various trials have reported the presence of pathological and misfolded proteins, in particular mutant huntingtin (mHtt) and phosphorylated tau neuropil threads, in the healthy grafted tissue. Here, we extended these observations with histological analysis of post-mortem tissue from three additional HD patients who had received similar striatal allografts from the fetal tissue transplantation trial conducted in Los Angeles in 1998. Immunohistochemical staining was performed using anti-mHtt antibodies, EM48 and MW7, as well as anti-hyperphosphorylated tau antibodies, AT8 and CP13. Immunofluorescence was used to assess the colocalization of EM48⁺ mHtt aggregates with the neuronal marker MAP2 and/or the extracellular matrix protein phosphacan in both the host and grafts. We confirmed the presence of mHtt aggregates within grafts of all three cases as well as tau neuropil threads in the grafts of two of the three transplanted HD patients. Phosphorylated tau was also variably expressed in the host cerebral cortex of all three subjects. While mHtt inclusions were present within neurons (immunofluorescence co-localization of MAP2 and EM48) as well as within the extracellular matrix of the host (immunofluorescence co-localization of phosphacan and EM48), their localization was limited to the extracellular matrix in the grafted tissue. This study corroborates previous findings that both mHtt and tau pathology can be found in the host and grafts of HD patients years post-grafting.

Keywords: Fetal neural transplantation; Huntington’s disease; Mutant huntingtin protein; tau.

Fig. 1.

Summary of patients’ characteristics. Information of all three transplanted HD patients. Light pink boxes represent new details and observations not published previously. Abbreviations: mHtt = mutant huntingtin; NFTs = neurofibrillary tangles; LC = left caudate; LP = left putamen; Ref = reference; RC = right caudate; RP = right putamen; # = number.

Fig. 2.

Histological evaluation of mHtt and phosphorylated tau within the host cortex and neuronal fetal allografts 8 years post-transplantation (case 1). Low power magnification image of one fetal tissue graft located in the putamen (A). Areas rich in striatal cells, referred to as p-zones, were further outlined (dotted line). Double immunohistochemical labelling revealed mHtt aggregates (Ni-DAB, dark purple to black) in the grafted tissue using both EM48 (B) and MW7 (C) antibodies (white arrows). As expected, mHtt aggregates (Ni-DAB, dark purple to black) were also detected in the host cortex using either EM48 (D) or MW7 (E) antibodies (white arrows) and quantifications were conducted in the host cortex, striatum and grafted tissue (F). Aggregates were further counted by volume size of either small (<0.75μm³) or medium/large (≥0.75μm³) (F). Phosphorylated tau (DAB brown) neuropil threads were detected in the graft using either AT8 (G) (phospho-tau Ser202 and Thr205) or CP13 (H) (pSer202) antibodies (black arrows). Phosphorylated tau (DAB brown) tangles and neuropil threads were observed in the host cortical tissue with AT8 (I) and CP13 (J) antibodies (black arrows). Scale bars: A = 1 cm; B-E, G-J = 20 μm. Abbreviations: ic = internal capsule; m = medium; MAP2 = microtubule-associated protein 2; mHtt = mutant huntingtin; pu = putamen; s = small; # = number.

Fig. 3.

Histological evaluation of mHtt and phosphorylated tau within the host cortex and neuronal fetal allografts 6 years post-transplantation (case 2). Low power magnification image of one fetal tissue graft located in the putamen (A). Areas rich in striatal cells, referred to as p-zones, were further outlined (dotted line). Double immunohistochemical labelling revealed mHtt aggregates (Ni-DAB, dark purple to black) in the grafted tissue using both EM48 (B) and MW7 (C) antibodies (white arrows). As expected, mHtt aggregates (Ni-DAB, dark purple to black) were also detected in the host cortex using either EM48 (D) or MW7 (E) antibodies (white arrows) and quantifications were conducted in the host cortex, striatum and grafted tissue (F). Aggregates were further counted by volume size of either small (<0.75μm³) or medium/large (≥0.75μm³) (F). Phosphorylated tau (DAB brown) neuropil threads were detected in the graft using either AT8 (G) (phospho-tau Ser202 and Thr205) or CP13 (H) (pSer202) antibodies (black arrows). Phosphorylated tau (DAB brown) neuropil threads were observed in the host cortical tissue with AT8 (I) and CP13 (J) antibodies (black arrows). Scale bars: A = 1 cm; B-E, G-J = 20 μm. Abbreviations: ic = internal capsule; m = medium; MAP2 = microtubule-associated protein 2; mHtt = mutant huntingtin; pu = putamen; s = small; # = number.

Fig. 4.

Histological evaluation of mHtt and phosphorylated tau within host cortex and neuronal fetal allografts 10 years post-transplantation (case 3). Low power magnification image of one fetal tissue graft located in the caudate (A). Areas rich in striatal cells, referred to as p-zones, were further outlined (dotted line). Double immunohistochemical labelling revealed mHtt aggregates (Ni-DAB, dark purple to black) in the grafted tissue using both EM48 (B) and MW7 (C) antibodies (white arrows). As expected, mHtt aggregates (Ni-DAB, dark purple to black) were also detected in the host cortex using either EM48 (D) or MW7 (E) antibodies (white arrows) and quantifications were conducted in the host cortex, striatum and grafted tissue (F). Aggregates were further counted by volume size of either small (<0.75μm³) or medium/large (≥0.75μm³) (F). Phosphorylated tau (DAB brown) neuropil threads were observed in the host cortical tissue using either AT8 (G) (phospho-tau Ser202 and Thr205) or CP13 (H) (pSer202) antibodies (black arrows). Scale bars: A = 1 cm; B-E, G-H = 20 μm. Abbreviations: ca = caudate; ic = internal capsule; m = medium; MAP2 = microtubule-associated protein 2; mHtt = mutant huntingtin; s = small; # = number.

Fig. 5.

Localization of EM48⁺ mHtt aggregates in the host cortical tissue of all three cases. Triple immunofluorescence labelling of EM48 (mHtt aggregates, green; white arrows), MAP2 (neuronal marker, red) and phosphacan (extracellular matrix, cyan) in HD host cortex of case 1 (A, A’), case 2 (B, B’) and case 3 (C, C’). Orthogonal views, from the selected area delineated with a dotted line in (A’, B’, C’), enabled the visualization of EM48⁺ mHtt aggregates within the extracellular matrix (phosphacan) as well as within neuronal elements (MAP2) (A”, B”, C”). Scale bars: A, A’, B, B’, C, C’ = 20 μm; A”, B”, C” = 5 μm. Abbreviations: MAP2 = microtubule-associated protein 2; mHtt = mutant huntingtin.

Fig. 6.

Localization of EM48⁺ mHtt aggregates in the grafted tissue of all three cases. Triple immunofluorescence labelling of EM48 (mHtt aggregates, green; white arrows), MAP2 (neuronal marker, red) and phosphacan (extracellular matrix, cyan) in the grafted tissue of case 1 (A, A’), case 2 (B, B’) and case 3 (C, C’). Orthogonal views, from the selected area delineated with a dotted line in (A’, B’, C’), enabled the visualization of EM48⁺ mHtt aggregates exclusively within the extracellular matrix (phosphacan), and not with the neuronal marker MAP2 (A”, B”, C”). Scale bars: A, A’, B, B’, C, C’ = 20 μm; A”, B”, C” = 5 μm. Abbreviations: MAP2 =, microtubule-associated protein 2; mHtt = mutant huntingtin.