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. 2015 Dec 20;24(25):7265-85.
doi: 10.1093/hmg/ddv426. Epub 2015 Oct 12.

A Role for Kalirin-7 in Corticostriatal Synaptic Dysfunction in Huntington's Disease

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Free PMC article

A Role for Kalirin-7 in Corticostriatal Synaptic Dysfunction in Huntington's Disease

Mar Puigdellívol et al. Hum Mol Genet. .
Free PMC article

Abstract

Cognitive dysfunction is an early clinical hallmark of Huntington's disease (HD) preceding the appearance of motor symptoms by several years. Neuronal dysfunction and altered corticostriatal connectivity have been postulated to be fundamental to explain these early disturbances. However, no treatments to attenuate cognitive changes have been successful: the reason may rely on the idea that the temporal sequence of pathological changes is as critical as the changes per se when new therapies are in development. To this aim, it becomes critical to use HD mouse models in which cognitive impairments appear prior to motor symptoms. In this study, we demonstrate procedural memory and motor learning deficits in two different HD mice and at ages preceding motor disturbances. These impairments are associated with altered corticostriatal long-term potentiation (LTP) and specific reduction of dendritic spine density and postsynaptic density (PSD)-95 and spinophilin-positive clusters in the cortex of HD mice. As a potential mechanism, we described an early decrease of Kalirin-7 (Kal7), a guanine-nucleotide exchange factor for Rho-like small GTPases critical to maintain excitatory synapse, in the cortex of HD mice. Supporting a role for Kal7 in HD synaptic deficits, exogenous expression of Kal7 restores the reduction of excitatory synapses in HD cortical cultures. Altogether, our results suggest that cortical dysfunction precedes striatal disturbances in HD and underlie early corticostriatal LTP and cognitive defects. Moreover, we identified diminished Kal7 as a key contributor to HD cortical alterations, placing Kal7 as a molecular target for future therapies aimed to restore corticostriatal function in HD.

Figures

Figure 1.
Figure 1.
HdhQ7/Q111 knock-in mutant mice display impaired learning of new motor skills at early disease stages when no motor coordination deficits are observed. Latency to fall in the accelerating rotarod task for WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mutant mice at 1 (A), 2 (B), 6 (C) and 8 (D) months of age. An age-dependent impairment in motor learning was observed in HD mice. Data represent the mean ± SEM (n = 9–14 per genotype). Statistical analysis was performed using two-way ANOVA with repeated measures. *P < 0.05; **P < 0.01; ***P < 0.001. Number of falls in the fixed rotarod at 10 and 25 rpm in WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mutant mice at 2 (E), 6 (F) and 8 (G) months of age. No motor coordination deficits were observed until 8 months of age in HdhQ7/Q111 mice. Data represent the mean ± SEM (n = 9–15 per genotype). Statistical analysis was performed using one-way ANOVA with post hoc Bonferroni's multiple comparison test. *P < 0.05.
Figure 2.
Figure 2.
R6/1 mice exhibit impaired learning of new motor skills at early disease stages. Latency to fall in the accelerating rotarod task for WT and R6/1 mice at 1 (A), 2 (B) and 3 (C) months of age. An age-dependent impairment in motor learning was observed in R6/1 mice. Data represent the mean ± SEM (n = 9–14 per genotype). Statistical analysis was performed using two-way ANOVA with repeated measures. *P < 0.05; **P < 0.01.
Figure 3.
Figure 3.
HdhQ7/Q111 and R6/1 mice display early impairments in procedural memory. Latency to reach the platform and error trials in the Swimming T-maze test in 6-month-old WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mice and in 2-month-old WT and R6/1 mice during the acquisition (A, C, E and G) and reversal (B, D, F and H) phases of the Swimming T-maze test of strategy shifting. HD mice exhibited procedural memory deficits in the reversal phase of the Swimming T-maze test. Data represent the mean ± SEM (n = 11–19 per genotype). Statistical analysis was performed using two-way ANOVA with repeated measures to analyze the latency to reach the platform: *P < 0.05; **P < 0.01. Logistic regression analysis using the Wald statistical test from IBM SPSS Statistics was used to analyze the error probability to reach the platform in the correct arm: *P < 0.05; **P < 0.01.
Figure 4.
Figure 4.
Abnormal corticostriatal synaptic plasticity and altered cortical dendritic spines in HdhQ7/Q111 mice at 2 months of age. (A) Summary data showing the time course of mean PS slope in WT HdhQ7/Q7 (open circle, n = 7) and knock-in mutant HdhQ7/Q111 (filled circle, n = 8) mice at 2–3 months of age in basal conditions and following LTP induction (arrows). For each slice, data were normalized to the average slope recorded during baseline. Data represent the mean ± SEM. Statistical differences, compared with pre-tetanus baseline amplitude values, were established using Student's two-tailed t test. ***P < 0.001. (B) (Left) Representative apical dendrites of the cortical pyramidal neurons of the motor cortex and dendrites of striatal medium spiny neurons of dorsal striatum from WT HdhQ7/Q7 and knock-in mutant HdhQ7/Q111 mice. Quantitative analysis showing dendritic spine density per micrometer of dendritic length. Knock-in HdhQ7/Q111 mice exhibit a significant reduction in cortical but not striatal dendritic spines. One-way ANOVA with Tukey post hoc comparisons was performed (63–83 dendrites; n = 4 animals per genotype); **P < 0.01 compared with HdhQ7/Q7 mice. (Right) Percentage of each morphological type of dendritic spine (see Materials and Methods for classification criteria) from WT HdhQ7/Q7 and knock-in mutant HdhQ7/Q111 mice at 2 months of age. One-way ANOVA with Tukey post hoc comparisons was performed (cortex: 319 spines from 30 dendrites from 4 animals per genotype; striatum: 280 spines from 25 dendrites from 4 animals per genotype were analyzed); *P < 0.01 (mushrooms versus thin spines).
Figure 5.
Figure 5.
Spinophilin-immunoreactive puncta are reduced in the motor cortex but not in the striatum of HdhQ7/Q111 mice. Representative confocal images showing spinophilin (red) positive clusters in the motor cortex (A and B) and dorsal striatum (C and D) of WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mice at 2 (A and C) and 8 (B and D) months of age. Spinophilin-immunoreactive puncta were counted and mean size evaluated in layers I, II/III and V of motor cortex area 1 (M1) and in the DL and DM striatum. Quantitative analysis is shown as mean ± SEM (n = 5–6 animals per group). A specific reduction in spinophilin-immunoreactive puncta was found in the cortex but not in the striatum of knock-in HdhQ7/Q111 mice from early disease stages. Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01 compared with HdhQ7/Q7 mice. Scale bar, 5 µm.
Figure 6.
Figure 6.
PSD-95-immunoreactive puncta are decreased at early disease stages in the motor cortex of HdhQ7/Q111 mice. Representative confocal images showing PSD-95 (green) positive clusters in the motor cortex (A and B) and dorsal striatum (C and D) of WT HdhQ7/Q7 and mutant knock-in HdhQ7/Q111 mice at 2 (A and C) and 8 (B and D) months of age. Cortical PSD-95-immunoreactive puncta were counted and mean size evaluated in layers I, II/III and V of motor cortex area 1 (M1) and in the DL and DM striatum. Quantitative analysis is shown as mean ± SEM (n = 5–6 animals per group). A specific reduction in PSD-95-immunoreactive puncta was found in cortex but not striatum of HdhQ7/Q111 mice at early disease stages. At more advanced disease stages, a reduction in PSD-95-immunoreactive puncta was also found in the dorsal striatum. Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with HdhQ7/Q7 mice. Scale bar, 5 µm.
Figure 7.
Figure 7.
Levels of Kal7 are reduced in the cortex but not in the striatum of HdhQ7/Q111 mice at early disease stages. Representative western blots showing the indicated subunits of glutamate receptors, pre- and postsynaptic scaffolding proteins and protein kinases in the total cortical (A) and striatal (B) extracts from 2- and 8-month-old WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mutant mice. α-Tubulin was used as a loading control. A specific reduction in Kal7 was found in the cortex but not in the striatum of HdhQ7/Q111 mutant mice compared with HdhQ7/Q7 WT mice at 2 months of age. At more advanced disease stages, a reduction in other synaptic-related proteins was found. Histograms represent the mean ± SEM and are expressed as percentage of WT animals (n = 5–8 animals per genotype). Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01 compared with HdhQ7/Q7 mice.
Figure 8.
Figure 8.
Kal7 levels are reduced in the cortex of R6/1 mice and in the cortex and putamen of HD brains. (A) Representative western blots showing Kal7 and α-tubulin as a loading control in total cortical and striatal extracts from 2- and 3-month-old WT and R6/1 mice. Histograms represent the mean ± SEM (n = 5–8 animals per genotype). Statistical analysis was performed using Student's two-tailed t test. **P < 0.01; ***P < 0.001 compared with HdhQ7/Q7 mice. (B) Representative western blots showing Kal7, GluN1, GluA1 and α-tubulin as a loading control in total cortical and putamen extracts from control (n = 4–7) and HD samples (n = 5–7). Histograms represent mean ± SEM and are expressed as percentage of control samples. Student's two-tailed t test was performed. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control human samples.
Figure 9.
Figure 9.
Rac1 activity is reduced in the cortex of HdhQ7/Q111 mice. Representative western blot of cortical extracts isolated from WT HdhQ7/Q7 and mutant HdhQ7/Q111 mice at 8 months of age showing Rac1-GTP and total Rac1 levels. Activated (GTP-bound) Rac1 was detected by immunoblotting of pull-down experiments from cortical extracts. Diminished Rac1 activity was found in HdhQ7/Q111 mutant mice compared with HdhQ7/Q7 WT mice (n = 5 animals per genotype), whereas similar total Rac1 levels were found between genotypes. Histograms represent mean ± SEM and are expressed as percentage of WT animals. Student's two-tailed t test was performed. *P < 0.05 compared with HdhQ7/Q7 mice.
Figure 10.
Figure 10.
Reduced levels of Kal7 associates with decreased number of synapse in mature cortical neurons from R6/1 mice. (A) Representative western blot showing Kal7 and α-actin as loading control in cortical neurons from WT and R6/1 mice at DIV14 (n = 3–8 animals per genotype) and DIV28 (n = 7–9 animals per genotype). A significant reduction in Kal7 levels was found in mutant R6/1 cortical neurons compared with WT cortical neurons at both DIV stages. Statistical analysis was performed using Student's two-tailed t test. *P < 0.05 compared with WT cortical neurons. (B and C) Representative confocal images showing Vglut1, PSD-95 and Vglut1 (red)/PSD-95 (green) positive clusters in WT and R6/1 cortical neurons. Cortical cultures were prepared from E18.5 WT (n = 3) and R6/1 (n = 5) embryos and triplicate cultures were fixed at DIV14 (B) and DIV28 (C). Clusters of Vglut1 and PSD-95 staining were quantified along the dendrite, and the number of excitatory synapses was measured as Vglut1/PSD-95 positive clusters. Differences in the number of excitatory synapses between WT and HD neurons were only detected at DIV28 (C) but not at DIV14 (B). Quantitative analysis of Vglut1, PSD-95 and Vglut1/PSD-95 positive clusters is shown as mean ± SEM [DIV14: n = 89 neurons (two dendrites/each neuron) from three WT mice (three replicates/animal); n = 149 neurons (two dendrites/each neuron) from five R6/1 mice (three replicates/animal); DIV28: n = 65 neurons (two dendrites/each neuron) from three WT mice (three replicates/animal); n = 89 neurons (two dendrites/each neuron) from five R6/1 mice (three replicates/animal)]. Scale bar, 10 µm. ***P < 0.01.
Figure 11.
Figure 11.
Exogenous expression of Kal7 restores the number of excitatory synapses in HD mature cortical neurons. Representative confocal images showing Vglut1 (red), PSD-95 (green) and Vglut1/PSD-95 positive clusters in WT and R6/1 cortical neurons transfected with Myc-His (MYC) or Myc-His-Kal7 (MYC-K7) vectors. Quantitative analysis of Vglut1, PSD-95 and Vglut1/PSD-95 positive clusters is shown as mean ± SEM [n = 65 neurons (two dendrites/each neuron) from three WT mice (three replicates/animal); n = 87–89 neurons (two dendrites/each neuron) from four R6/1 mice (three replicates/animal)]. Overexpression of Myc-K7 restores the number of PSD-95, Vglut1 and Vglut1/PSD95 positive clusters (excitatory synapses) in R6/1 cortical neurons. Statistical analysis was performed using one-way ANOVA with post hoc Bonferroni's multiple comparison test. ***P < 0.001 compared with WT Myc. ###P < 0.001 compared with the corresponding (WT or R6/1) Myc-control.

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