Fyn Kinase-Mediated PKCδ Y311 Phosphorylation Induces Dopaminergic Degeneration in Cell Culture and Animal Models: Implications for the Identification of a New Pharmacological Target for Parkinson's Disease

Abstract

Oxidative stress, neuroinflammation and apoptosis are some of the key etiological factors responsible for dopamin(DA)ergic degeneration during Parkinson's disease (PD), yet the downstream molecular mechanisms underlying neurodegeneration are largely unknown. Recently, a genome-wide association study revealed the FYN gene to be associated with PD, suggesting that Fyn kinase could be a pharmacological target for PD. In this study, we report that Fyn-mediated PKCδ tyrosine (Y311) phosphorylation is a key event preceding its proteolytic activation in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinsonism. MPP⁺/MPTP induced Fyn kinase activation in N27 DAergic neuronal cells and the mouse substantia nigra. PKCδ-Y311 phosphorylation by activated Fyn initiates the apoptotic caspase-signaling cascade during DAergic degeneration. Pharmacological attenuation of Fyn activity protected DAergic neurons from MPP⁺-induced degeneration in primary mesencephalic neuronal cultures. We further employed Fyn wild-type and Fyn knockout (KO) mice to confirm whether Fyn is a valid pharmacological target of DAergic neurodegeneration. Primary mesencephalic neurons from Fyn KO mice were greatly protected from MPP⁺-induced DAergic cell death, neurite loss and DA reuptake loss. Furthermore, Fyn KO mice were significantly protected from MPTP-induced PKCδ-Y311 phosphorylation, behavioral deficits and nigral DAergic degeneration. This study thus unveils a mechanism by which Fyn regulates PKCδ's pro-apoptotic function and DAergic degeneration. Pharmacological inhibitors directed at Fyn activation could prove to be a novel therapeutic target in the delay or halting of selective DAergic degeneration during PD.

Keywords: Fyn; PKCδ; kinase; neurodegeneration; phosphorylation.

FIGURE 1

Activated Fyn kinase phosphorylates PKCδ Tyr 311 in N27 dopaminergic neuronal cells during MPP⁺ treatment. N27 cells were exposed to 300 μM MPP⁺ for up to 24 h. (A) Fyn kinase activity assay of cell lysates that were incubated with a Fyn kinase-specific substrate following 3 h treatment with MPP⁺. (B) Western blot and densitometric analysis showing time-dependent Y311 phosphorylation of PKCδ in MPP⁺-treated N27 cells. Representative Western blot images from 3 independent experiments are shown. (C) Western blot and densitometric analysis showing the effects of MPP⁺ at the 3-h time-point on protein expression levels of Fyn, PKCδ and p-Y311-PKCδ in N27 cells transiently transfected with Fyn-specific siRNA or non-specific (NS) siRNA. (D) Caspase-3 activation assay of the transfected cells described above at 24 h time-point. Data are represented as mean ± SEM from three independent experiments. Statistically significant treatment differences indicated by **p < 0.01, ***p < 0.001 when comparing control with MPP⁺; and ^# p ≤ 0.05, ^### p < 0.001 when comparing Fyn and non-specific siRNA for the MPP⁺ treatments, n = 3–4 per group.

FIGURE 2

TSKI attenuates MPP⁺-induced dopaminergic neuronal degeneration in mesencephalic primary cultures. (A) Immunofluorescence images showing discernible attenuation of neurodegeneration in primary mesencephalic neurons exposed to 5 μM MPP⁺ for 48 h in the presence or absence of 5 μM TSKI. (B) MetaMorph image analysis of neuronal process lengths of the primary mesencephalic neurons described above. (C) MetaMorph image analysis of TH⁺ cell count. Statistically significant treatment differences indicated by **p < 0.01 for Control v. MPP⁺ and ^## p < 0.01 MPP⁺ v. MPP⁺ + TSKI (n = 4).

FIGURE 3

Fyn-deficient primary dopaminergic neurons are protected against MPP⁺-induced neurodegeneration. (A) Immunofluorescence images showing Fyn kinase (in red), tyrosine hydroxylase (in green) and nuclei (in blue) of primary mesencephalic neurons cultured from Fyn wild-type (WT) and knockout (KO) mice exposed to 3 or 5 μM MPP⁺ for 48 h. (B, C) MetaMorph image analysis of (B) TH⁺ cell count and (C) neuron process lengths in WT and Fyn KO primary mesencephalic cultures 48 h after exposure to 5 μM MPP⁺. D, High-affinity [³H] dopamine uptake by Fyn WT and KO midbrain primary neurons after exposure to 3 and 5 μM MPP⁺. Statistically significant treatment differences indicated by **p < 0.01, ***p < 0.001 when comparing control with MPP⁺; and ^# p ≤ 0.05, ^## p < 0.01, ^### p < 0.001 when comparing Fyn WT and KO for the MPP⁺ treatments, n = 4 per group.

FIGURE 4

MPTP induces Fyn kinase activity and PKCδ Tyr 311 phosphorylation in mouse substantia nigra (SNc). (A) Western blot of SNc tissue lysates from mice 24 h after injection with vehicle or MPTP. Representative Western blot images from 3 independent experiments are shown. (B) Densitometric analysis of Western blot of p-Y311-PKCδ. (C) Fyn kinase activity assay of SNc tissue lysates incubated with a Fyn kinase-specific substrate. (D) Immunofluorescence images of TH-Fyn double immunostaining revealing that MPTP induces Fyn kinase expression in TH⁺ neurons. Data represent mean ± SEM from n = 3–6 mice per group. Statistically significant treatment differences indicated by **p < 0.01, ***p < 0.001; n = 4–6 mice per group.

FIGURE 5

Fyn deficiency suppresses MPTP-induced locomotor deficits. Wild-type (WT) and Fyn knockout (KO) mice were injected with saline or MPTP and were then euthanized on day 7 after open-field and Rota-Rod performance was measured on days 5 and 6. (A) Track of horizontal (lines) and vertical (dots) movements. (B) Total horizontal and (C) vertical activities. (D) Total distance moved. (E) Movement time. (F) Total number of discrete horizontal movements. (G) Stereotypy count. (H) Number of stereotypy movements (bouts or episodes) and (I) Average time spent on Rota-Rod. Data are represented as mean ± SEM from 6 to 8 animals per group. Statistically significant treatment differences indicated by *p ≤ 0.05, **p < 0.01, ***p < 0.001 for WT control v. WT MPTP groups; and ^# p ≤ 0.05, ^## p < 0.01 for WT MPTP v. KO MPTP groups.

FIGURE 6

Fyn kinase deficiency attenuates MPTP-induced depletion of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). (A)–(C) Neurochemical analysis via HPLC of striatal tissue lysates from wild-type (WT) and Fyn knockout (KO) mice 7 days post saline or MPTP treatment. (A) DA. (B) HVA. (C) DOPAC. Data are represented as mean ± SEM from n = 10-12 mice per group. Statistically significant treatment differences indicated by ***p < 0.001 for WT control v. WT MPTP groups; and ^## p < 0.01, ^### p < 0.001 for WT MPTP vs. KO MPTP groups.

FIGURE 7

Fyn deficiency protects mice from MPTP-induced dopaminergic neurodegeneration. (A) Western blot of Fyn kinase and TH protein expression in SNpc lysates from wild-type (WT) and Fyn knockout (KO) mice post saline or MPTP treatment. Densitometric analysis of the immunoblots in 7A with relative densities of TH normalized to the densities of the corresponding β-Actin signals following 7 days post saline or MPTP treatment. Values (in arbitrary units) for TH are expressed as mean ± SEM for at least 6–8 mice per treatment. (B) Western blot of Fyn kinase and p-Tyr-311-PKCδ protein expression from the SNc following 24 h post saline or MPTP treatment. Densitometric analysis of the immunoblots normalized against β-actin. Statistically significant treatment differences indicated by **p < 0.01 for WT control v. WT MPTP groups; and ^# p ≤ 0.05 for WT MPTP v. KO MPTP groups.

FIGURE 8

Fyn deficiency protects against the loss of substantia nigral (SNc) TH⁺ neurons and neurites in MPTP-treated mice. (A) Immunoperoxidase (DAB) staining of striatal TH sections from wild-type (WT) and Fyn knockout (KO) mice 7 days after the last MPTP injection. (B) TH DAB staining of SNc. (C) Total count of TH⁺ neurons in the SNc of groups exposed to either saline or MPTP, measured using unbiased stereology. Data are represented as mean ± SEM from n = 4 mice per group. Statistically significant treatment differences indicated by ***p < 0.001 for WT control v. WT MPTP groups; and ^### p < 0.001 for WT MPTP v. KO MPTP groups.

FIGURE 9

The role of Fyn kinase in mediating cell death signaling in Parkinson’s disease. The Parkinsonian toxicant MPTP’s active metabolite MPP⁺ produces mitochondrial dysfunction to generate reactive oxygen species (ROS) that trigger the activation of Fyn kinase. The early stress sensor molecule Fyn further phosphorylates PKCδ on the tyrosine 311 residue flanking the caspase-3 recognition site. This serves as the priming signaling event that allows caspase-3 to proteolytically activate PKCδ to initiate molecular events that cause dopaminergic cell death. The constitutively active catalytic fragment (CF) of PKCδ further amplifies the dopaminergic cell death events by feedback activation of caspase-3.