Down-regulation of neuroprotective protein kinase D in Huntington´s disease

Abstract

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder characterized by the selective dysfunction and loss of neurons in the striatum and cerebral cortex. Experimental evidence suggests that GABAergic medium-sized spiny neurons (MSNs) in the striatum are particularly vulnerable to glutamate-induced toxicity (excitotoxicity) and its analogues. However, the molecular mechanisms underlying MSN-specific death in HD remain poorly understood. The serine/threonine protein kinase D1 (PKD1) confers neuroprotection in various neuropathological conditions, including ischemic stroke. While excitotoxicity inactivates PKD1 in cortical glutamatergic neurons without altering its levels, active PKD1 potentiates the survival of excitatory neurons in highly excitotoxic environments. Here, we investigated whether PKD1 activity dysregulation contributes to MSN death in HD and its association with neurodegeneration. We found an unexpected reduction in PKD1 protein levels in striatal neurons from HD patients. Similarly, the R6/1 mouse model of HD exhibited progressive PKD1 protein loss, commencing at early disease stages, accompanied by decreased Prkd1 transcript levels. PKD1 downregulation also occurred in the cerebral cortex of R6/1 mice, but only at late stages. Functionally, pharmacological PKD inhibition in primary striatal neurons exacerbated excitotoxic damage and apoptosis induced by glutamate N-methyl D-aspartate (NMDA) receptors, whereas expression of constitutively active PKD1 (PKD1-Ca) conferred neuroprotection. Furthermore, PKD1-Ca protected against polyQ-induced apoptosis in a cellular model of HD. In a translational approach, intrastriatal lentiviral delivery of PKD1-Ca in symptomatic R6/1 mice prevented the loss of DARPP-32, a molecular marker of MSNs. Collectively, our findings strongly suggest that PKD1 loss-of-function contributes to HD pathogenesis and the selective vulnerability of MSNs. These findings position PKD1 as a promising therapeutic target for mitigating MSN death in HD.

Fig. 1. Decreased neuronal PKD levels in striatum from HD patients.

A Representative confocal microscopy images of PKD_T staining (red channel) in neurons (NeuN⁺, green channel, full arrowhead) and astrocytes (GFAP⁺, purple channel, empty arrowhead) in striatum from HD patients and control individuals. Scale bar: 40 µm (top panels) and 20 µm (bottom panels). The top graph represents the quantification of PKD_T intensity staining in striatal neurons of control individuals compared to HD patients (n = 3 individuals per group). The bottom graph shows the quantification of PKD_T staining intensity in striatal astrocytes from control individuals compared to HD patients (21 and 58 GFAP-positive striatal astrocytes in control and HD samples, respectively). Nuclei were stained with DAPI. B PKD_T immunohistochemistry in cerebral cortex sections from HD patients and control individuals. Each panel is a representative image from different individuals Scale bar: 100 µm. Chromogen detection was quantified and expressed as the percentage of area stained by PKD_T from sections (n = 4 individuals per group). C Representative immunoblot of PKD_T and quantification of protein levels in homogenates from striatum necropsies from HD patients (n = 6) and control non-affected individuals (n = 6). Levels of β-actin were used as loading control for normalization purposes. D qRT-PCR analysis of PRKD1 mRNA from striatum and cortex of HD patients (n = 6–8) and non-affected individuals (n = 5–9). Data are represented as mean ± SEM. *P < 0.05 or n.s. (not significant) using unpaired Student’s t test.

Fig. 2. Early loss of striatal PKD in R6/1 mouse model of HD.

Representative immunoblot of PKD_T and p-PKD (S⁹¹⁶) and quantification of protein levels in homogenates from striatum and cerebral cortex of 3.5- (A) or 7.5-month-old (B) R6/1 (n = 6) and wild type (WT) mice (n = 7) of the same ages. Levels of β-actin were used as loading control for normalization purposes. C qRT-PCR analysis of PRKD1 mRNA from striatum of 3.5-month-old or 7.5-month-old R6/1 (n = 6–7) and WT (n = 6–7) mice. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 or ***P < 0.001 using unpaired Student’s t test.

Fig. 3. Early loss of striatal neuronal PKD in R6/1 mice occurs mainly in neurons while PKD expression appears in astrocytes.

A Representative confocal microscopy images of PKD_T (red channel) in neurons (NeuN⁺, green channel) in striatum of 3.5-month-old WT and R6/1 mice. Nuclei were stained with DAPI. Scale bar: 20 µm. Graph represents the quantification of PKD_T intensity staining in striatal neurons of WT compared to R6/1 mice (3 animals per group). PKD_T immunohistochemistry in cerebral cortex (B) and globus pallidum (C) sections from 7.5-month-old WT and R6/1 mice. Scale bar: 150 µm. Graphs represent the quantification of chromogen detection and was expressed as DAB-staining reciprocal intensity (Arbitrary Units, A. U.) (B) or quantification of PKD_T-positive astrocytes per area (C) (4 animals per group). Data are represented as mean ± SEM. * P < 0.05 or ** P < 0.01 using unpaired Student’s t test.

Fig. 4. PKD inhibition enhances excitotoxicity-induced DARPP-32 loss in cultured rat primary striatal neurons by mechanisms dependent on NMDARs, calcium and calpain.

PKD_T, p-PKD (S⁹¹⁶) and DARPP-32 immunoblot analysis of cultured primary mature striatal neurons stimulated with excitotoxic concentrations of NMDA (50 µM) plus its co-agonist glycine (10 µM) (a treatment referred hereafter as “NMDA”) for the indicated times (A–C), in combination with the PKD-specific pharmacological inhibitor CRT0066101 (CRT, 5 µM) 1 h before NMDA-treatment (B, C) or together with the calpain inhibitor Ci-III (20 µM) (C) and remained in the culture media for the duration of the experiment. Spectrin full-length (FL) and break-down products (BDPs) confirming excitotoxicity and calpain activation are also shown. Graphs represent the quantification of PKD_T, p-PKD (S⁹¹⁶), spectrin BDPs (A, B) and DARPP-32 (A–C) immunoblot signal relative to loading control neural-specific enolase (NSE). n = 3–4 independent experiments. D PKD_T, p-PKD (S⁹¹⁶) and DARPP-32 immunoblot analysis of cultured striatal neurons pre-treated for 1 h with the calpain inhibitor Ci-III, the NMDAR antagonist DL-AP5 or the Ca²⁺ chelator EGTA, and then stimulated with NMDA for 2 h. Graph represents the quantification of DARPP-32 immunoblot signal relative to loading control NSE. n = 3 independent experiments. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 or ****P < 0.0001 using one-way ANOVA, followed by Bonferroni’s post hoc test.

Fig. 5. PKD inhibition enhances excitotoxic cell death of cultured striatal neurons.

A Representative confocal microscopy images of cultured primary mature striatal neurons (MAP2⁺ cells, green channel) stimulated with NMDA in combination with CRT for the indicated times. Nuclei were stained with DAPI. Scale bar: 40 µm. Graphs represents the percentage of area covered by MAP2 staining and the percentage of condensed nuclei (n = 3 independent experiments). B Representative fluorescence microscopy (top panels) and corresponding brightfield images (bottom panels) of cultured primary mature striatal neurons stimulated with NMDA in combination with CRT for the indicated times. After treatment, neurons were stained with Annexin V-FITC (AxV) and Propidium Iodide (PI). Scale bar: 20 µm. The graph represents the percentage of early apoptotic neurons (AxV⁺/PI^-) relative to the total number of neurons in each condition (n = 3 independent experiments). All data are represented as mean ± SEM. *P < 0.05, **P < 0.01 or ***P < 0.001 using one-way ANOVA, followed by Bonferroni’s post hoc test.

Fig. 6. Neuroprotection of cultured striatal neurons against NMDA-induced excitotoxicity by PKD1.

A Scheme of the vector construct used for neuronal expression of a constitutively active PKD1 mutant (PKD1-Ca). GFP alone or fused to PKD1-Ca was cloned under the neurospecific human synapsin promoter (hSYN). B Representative confocal microscopy images of cultured rat primary mature striatal neuron transfected with GFP (Control) or PKD1-Ca and stained with p-PKD (S⁹¹⁶) and DAPI. Scale bar: 25 µm. C Representative confocal microscopy images of GFP, DARPP-32 and DAPI signal of neurons transduced with lentiviral particles for GFP and GFP-PKD1-Ca expression treated or not with NMDA for 2 h. Scale bar: 40 µm. Top Graph represents the percentage of DARPP-32⁺ surviving neurons after NMDA treatment, relative to total GFP⁺ neurons in untreated conditions. Bottom graph represents the percentage of GFP⁺ neurons bearing condensed nuclei relative to total GFP⁺ neurons (n = 20–60 neurons per condition; n = 4 independent experiments). All data are represented as mean ± SEM. *P < 0.05, **P < 0.01 or ***P < 0.001 using one-way ANOVA, followed by Bonferroni’s post hoc test.

Fig. 7. PKD1 protects from polyglutamine-induced apoptosis in a cell model of HD.

A Representative immunoblot of PKD_T and p-PKD (S⁹¹⁶) in homogenates from neuroblastoma N2a cell line transfected with PolyQ16 or PolyQ94 in combination with p-EF-BOS-GFP (Ctrl) or p-EF-BOS-PKD1-Ca (PKD1-Ca). Levels of β-actin were used as loading control. B Representative confocal microscopy images of GFP, CFP, and cleaved caspase-3 in the neuroblastoma N2a cell line transfected as described in (A). Nuclei were stained with To-Pro. Scale bar: 10 µm. Graph represents the quantification of cleaved caspase-3⁺ cells relative to GFP/CFP⁺ cells 24 h post-transfection (n = 25 cells per condition; n = 3 per group). All data are represented as mean ± SEM. **P < 0.01, using one-way ANOVA, followed by Bonferroni’s post-hoc test.

Fig. 8. In vivo expression of GFP-PKD1-Ca enhances DARPP-32 levels in R6/1 striatum.

Panels show representative confocal microscopy images of GFP, DARPP-32 and DAPI signal of contralateral and ipsilateral R6/1 striatum of 6-month-old mice injected with GFP-PKD1-Ca lentiviral particles. Graph represents normalized DARPP-32 mean intensity of GFP-PKD1-Ca transduced cells, the equivalent contralateral area and GFP-PKD1-Ca non-expressing neurons adjacent to transduced cells. All data are represented as mean ± SEM. ***P < 0.001, using one-way ANOVA, followed by Bonferroni’s post-hoc test.