Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex

Abstract

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

Keywords: Huntington disease; cortical neurogenesis; mitosis; spindle orientation.

Figure 1.

mHTT causes spindle misorientation. A, Measurement of the spindle angle α. STHdh^Q7/Q7 (Q7) and STHdh^Q111/Q111 (Q111) cells stained as indicated. B, C, Spindle angles of Q7, Q111 (B), and HeLa (C) cells. D, E, Spindle angles of Q7 (D) and Q111 (E) cells grown on micropatterns. Time-lapse pictures were used to measure the positioning of the metaphasic plate (H2B-mCherry). Scale bars, 10 μm. Error bars indicate SEM. **p < 0.01 (unpaired, one-tailed t test). ***p < 0.001 (unpaired, one-tailed t test).

Figure 2.

mHTT alters the mitotic distribution of p150^Glued, NuMA, and Dynein. A, STHdh^Q7/Q7 (Q7) and STHdh^Q111/Q111 (Q111) cells were stained as indicated. Scale bars, 10 μm. B, Representative line scan analysis showing the distribution of p150^Glued and NuMA. C, Percentage of cells with cortical labeling of p150^Glued and NuMA. D, Percentage of Q7 and Q111 cells with dynein on astral microtubules. C, D, At least n = 25 cells in three independent experiments. Error bars indicate SEM. **p < 0.01 (unpaired, one-tailed t test). ***p < 0.001 (unpaired, one-tailed t test).

Figure 3.

+TIPs localization at microtubules plus-ends and microtubule dynamics are modified in HD. A, C, Immunostainings of EB3-GFP transfected STHdh^Q7/Q7 (Q7) and STHdh^Q111/Q111 (Q111) cells with anti-CLIP170 (A) and anti-p150^Glued (C) antibodies. N, nucleus. B, D, Representative line scan analysis of fluorescence intensity of CLIP170 (B) or p150^Glued (D), and EB3, starting at the end of the MT to 3 μm inwards. E, Time-lapse recordings illustrating the MT dynamic instability in Hdh^Q7/Q7 (MEF Q7) and Hdh^Q111/Q111 (MEF Q111) MEFs expressing mCherry-α-tubulin and EB3-GFP. Arrowheads indicate localization of the MT tip close to the leading edge. Time is expressed as min:sec. F–H, MT dynamics in Q7 and Q111 MEFs (at least 5 MTs examined in n = 10 cells in two independent experiments). F, Mean velocity of EB3-comets. G, Persistence time. H, MT catastrophe rate. Scale bars, 10 μm. Error bars indicate SEM. NS, Not significant. **p < 0.01 (unpaired, one-tailed t test). ***p < 0.001 (unpaired, one-tailed t test).

Figure 4.

Spindle orientation of dividing progenitors and cerebral cortex thickness are altered in Hdh^Q111/Q111 embryos. A, Illustration of dividing progenitors. White dashed line indicates spindle; yellow line indicates ventricular surface. γ-tubulin marks centrosomes. Scale bar, 15 μm. B, C, Spindle angles during divisions in E10.5 (B) and E14.5 (C) embryos at metaphase (meta) and anaphase (ana). D, Nestin and β-III-tubulin immunostainings of E14.5 coronal sections. Scale bars, 50 μm. E, F, VZ, IZ, CP (E) and overall cortical (F) thicknesses (n = 3 embryos per genotype). G, H, Tbr1 (G) and Cux1 (H) immunostainings of P21 coronal sections. Scale bars, 100 μm. I, Layers VI and II-IV thicknesses (n = 3 mice per genotype). J, K, Spindle angles of STHdh^Q7/Q7 (Q7), STHdh^Q111/Q111 (Q111), and STHdh^Q111/Q111 cells transfected with Akt c.a. (J) and of HeLa cells transfected with the indicated constructs (K). L, Spindle angles in E16.5 electroporated progenitors. Error bars indicate SEM. NS, Not significant. *p < 0.05 (unpaired, one-tailed t test). **p < 0.01 (unpaired, one-tailed t test). ***p < 0.001 (unpaired, one-tailed t test).