Alzheimer risk gene product Pyk2 suppresses tau phosphorylation and phenotypic effects of tauopathy

Tau and Pyk2 sequences were subcloned into an AAV-CAG-GFP vector (RRID:Addgene_28014) and GSK3β and Fyn sequences subcloned into a pcDNA3.0 vector which served as a negative transfection control.

Human embryonic kidney 293T (Hek293T, RRID:CVCL_QW54) cells were cultured in DMEM (Gibco #11965092) with 10% FBS (Gibco #16000044) and incubated at a constant 37 °C with 5% CO₂. For protein over-expression, Hek293T cells were transfected with appropriate DNA constructs (0.5 μg DNA/well in a 12-well plate) using Lipofectamine 3000 reagent (Invitrogen #L3000001). Cells were harvested in 1% Triton X-100 containing 50 mM Tris, 150 mM NaCl and 1 mM EDTA with protease (Roche #11836170001) and phosphatase (Roche #4906845001) inhibitors. Lysates were spun at 14,000×g for 10 min at 4 °C and Triton X-100-soluble supernatants were boiled in Laemmli sample buffer (Bio-Rad #1610747) at 95 °C for 10 min.

B6;C3-Tg (Prnp-MAPT*P301S) PS19Vle/J (RRID:IMSR_JAX:008169) mice purchased from Jackson Laboratories (JAX) were backcrossed over 5 generations to a C57BL/6 J background to obtain hemizygous PS19^0/+ animals. Pyk2^−/− mice, backcrossed to a C57BL/6J background over 10 generations by Schlessinger and colleagues [39] (RRID:MGI_3584536), were generously provided by Dr. David Schlaepfer (University of California–San Diego). Hemizygous PS19^0/+ and Pyk2^+/− mice were paired to obtain WT, Pyk2^−/−, PS19^0/+ and Pyk2^−/−; PS19^0/+ mice. All experiments used littermate control mice with no preference for either female or male animals. Comparisons of male and female outcomes by group were conducted post hoc. All protocols including animal husbandry, genotyping, behavioral testing and euthanasia were approved by the Yale University Institutional Animal Care and Use Committee (IACUC). Animals were housed in groups of 2–5 mice per cage with access to food and water ad libitum. 12-h light/dark cycles were maintained throughout the duration of animal housing with light periods beginning consistently at 7 am.

4.5–5.5-month-old PS19^0/+ mice were sacrificed via live decapitation in accordance with Yale University’s Institutional Animal Care and Use Committee standards. Brains were dissected on ice and sectioned using a Leica WT1000S Vibratome in ice-cold, oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid (aCSF) containing 119 mM NaCl, 2.5 mM KCl, 2.7 mM MgSO₄, 26 mM NaHCO₃, 11 mM D-glucose and 1.25 mM NaH₂PO₄. Three 400-μm-thick coronal sections (containing the three most rostral sections of hippocampus) were collected per brain. The two hemispheres of each section were divided medially and slices enriched for hippocampal tissue by removal of the ventral half of each section using a sharp razor blade. Hippocampal-enriched brain slices were transferred to a radial-arm incubation chamber (Scientific Systems Designs Inc. #BSK6–6) containing room temperature aCSF supplemented with 2.4 mM CaCl₂ and continuously oxygenated with 95% O₂ and 5% CO₂. After a 30 min recovery, slices were treated with either 1 μM PF-719 (Chinglu Pharmaceutical Research) or equal volume of DMSO for 2 h at room temperature. After treatment, slices were immediately homogenized on ice in 100 μl RIPA (EMD Millipore #20–188) with protease and phosphatase inhibitors (Thermo Scientific #1861281) using a polypropylene pellet pestle and spun at 100,000×g for 30 min at 4 °C. RIPA-soluble supernatants were boiled in Laemmli sample buffer with 5% β-mercaptoethanol and 1% SDS at 95 °C for 5 min.

9.5–10.5-month-old animals were sacrificed via live decapitation and hemispheres separated medially on ice using a sharp razor blade. For immunohistology, left hemispheres were post-fixed in PBS with 4% paraformaldehyde (PFA) for 48 h at 4 °C. Post-fixed hemispheres were then transferred to PBS with 0.05% sodium azide and stored at 4 °C until sectioning. For biochemistry, hippocampi and cortices were immediately dissected from right hemispheres on ice. To obtain TBS-soluble fractions, hippocampi and cortices were homogenized in 150 μl and 300 μl, respectively, TBS with protease and phosphatase inhibitors using a polypropylene pellet pestle on ice. Homogenates were spun at 100,000×g for 30 min at 4 °C. TBS-soluble supernatants were boiled in Laemmli sample buffer with 5% β-mercaptoethanol and 1% SDS at 95 °C for 5 min, while TBS-insoluble hippocampal and cortical pellets were resolubilized on ice in 150 μl and 300 μl, respectively, RIPA with 1% SDS and protease/phosphatase inhibitors. Homogenates were spun at 100,000×g for 30 min at 4 °C and SDS-soluble supernatant boiled in Laemmli sample buffer with 5% β-mercaptoethanol and 1% SDS at 95 °C for 5 min.

Samples were separated via SDS-PAGE through 4–20% Tris-glycine gels (Bio-Rad #5671095) and transferred onto nitrocellulose membranes (Invitrogen #IB23001) using an iBlot 2 Gel Transfer Device (Invitrogen #IB21001). Loaded sample volumes were normalized to total protein concentration via BCA protein assay (Thermo Scientific #23225). Nitrocellulose membranes were blocked while rocking at room temperature for 1 h (Rockland #MB-070-010TF) and incubated with blocking buffer containing primary antibodies overnight at 4 °C. The following antibodies were employed for immunoblotting: anti-Tau (HT7) (Thermo Fisher Scientific #MN1000, 1:1000, RRID:AB_2314654), anti-pTau S202/T205 (AT8) (Thermo Fisher Scientific #MN1020, 1:1000, RRID:AB_223647), anti-pTau S396/S404 (PHF-1) (Peter Davies personal request, 1:1000, RRID:AB_2315150), anti-pTau S199/S202 (Thermo Fisher Scientific #44-768G, 1:1000, RRID:AB_2533749), anti-pTau T181 (AT270) (Thermo Fisher Scientific #MN1050, 1:1000, RRID:AB_223651), anti-pTau S262 (Thermo Fisher Scientific #44-750G, 1:1000, RRID:AB_2533743), anti-Pyk2 (Cell Signaling Technology #3480, 1:1000, RRID:AB_2174093), anti-pPyk2 Y402 (Cell Signaling Technology #3291, 1:1000, RRID:AB_2300530), anti-GSK3β (Cell Signaling Technology #9315, 1:1000, RRID:AB_490890), anti-GSK3β Y216/pGSK3α Y279 (Abcam #ab68476, 1:1000, RRID:AB_10013745), anti-GSK3β S9 (Cell Signaling Technology #9336, 1:1000, RRID:AB_331405), anti-Fyn (Cell Signaling Technology #4023, 1:1000, RRID:AB_10698604), anti-pSrc Family Y216 (Cell Signaling Technology #6943, 1:1000, RRID:AB_10013641), anti-PSD-95 (Cell Signaling Technology #36233, 1:1000, RRID:AB_2721262), anti-C1q (Abcam #ab182451, 1:1000, RRID:AB_2732849), anti-p38 MAPK (Cell Signaling Technology #9212, 1:1000, RRID:AB_330713), anti-pp38 MAPK T180/Y182 (Cell Signaling Technology #4511, 1:1000, RRID:AB_2139682), anti-LKB1 (Cell Signaling Technology #3047, 1:1000, RRID:AB_2198327), anti-pLKB1 S428 (Cell Signaling Technology #3482, 1:1000, RRID:AB_2198321), anti-MAPK1 (Cell Signaling Technology #4696, RRID:AB_390780), anti-pMAPK1 T185/Y187 (Cell Signaling Technology #9101, 1:1000, RRID:AB_331646) and anti-β-Actin (Cell Signaling Technology #3700, 1:10,000, RRID:AB_2242334). For experiments shown in Fig. 1, all primary antibodies were the same as above accept for the following: anti-GSK3β (Cell Signaling Technology #12456, 1:2000, RRID:AB_2636978), anti-GSK3β Y216/pGSK3α Y279 (Abcam #ab52188, 1:1000, RRID:AB_880261), anti-Pyk2 (Cell Signaling Technology #3292, 1:1000, RRID:AB_2174097) and anti-β-Actin (Cell Signaling Technology #8457, 1:1000, RRID:AB_10950489). After primary antibody incubation, membranes were washed (3 X 5-min in TBST) and incubated with appropriate secondary antibodies for 1 h at room temperature. The following secondary antibodies were used for immunoblotting: anti-mouse IRDye 800CW (LI-COR Biosciences #926–32,212, 1:20,000, RRID:AB_621847) and α-rabbit IRDye 680CW (LI-COR Biosciences #926–68,023, 1:20,000, RRID:AB_10706167). After final washes (5 X 5-min in TBST), immunoblots were scanned with a LI-COR Odyssey infrared imaging system and protein bands quantified using LI-COR Image Studio Lite software, version 5.2.5 (RRID:SCR_013715).

Behavioral assays were administered in the following order: rotarod, wire hang and Morris Water maze (MWM). Noldus CatWalk XT gait analysis was administered to a separate cohort of animals naïve to rotarod, wire hang and MWM. All animals were 9–10 months old at the time of behavioral testing. Exclusion criteria, described below, were applied independently to each assay. Animal handling and analyses (including application of exclusion criteria) were completed by an experimenter blinded to animal genotype. In order to habituate animals to experimenter handling, mice were handled for at least 2 min over 4 days prior to the first behavioral assay. For each assay, mice were habituated to the testing room for 1 h prior to behavioral testing.

Animals were sacrificed and brain tissue dissected on ice as described above. Hippocampi and cortices from a single hemisphere were homogenized in 200 μl and 400 μl, respectively, Syn-PER Reagent (Thermo Scientific #87793) with protease and phosphatase inhibitors using a polypropylene pellet pestle. Homogenates were centrifuged at 1200×g for 10 min at 4 °C. Supernatants were collected and spun again at 15,000×g for 20 min at 4 °C. The crude synaptosomal (P2’) pellets were resolubilized in RIPA with 1% SDS and boiled in Laemmli sample buffer with 5% β-mercaptoethanol and 1% SDS at 95 °C for 5 min.

One-way ANOVA with post hoc Tukey’s multiple comparisons tests, One-way ANOVA with post hoc Dunnett’s multiple comparisons tests, Log-rank (Mantel-Cox) test and unpaired two-tailed t-tests were performed using GraphPad Prism software, versions 8 and 9 (RRID:SCR_002798). Repeated measures ANOVA with post hoc Tukey HSD multiple comparisons tests were performed using IBM SPSS Statics software, version 26 (RRID:SCR_019096). For the MWM visible platform test, statistical outliers were identified using the ROUT method (Q = 1%) in GraphPad Prism. Group means ± SEM and samples sizes (n) are reported in each figure legend. Data were considered to be statistically significant if p < 0.05. For all figures, all statistically significant group differences are labeled. For any given group comparison, the lack of any indication of significant difference implies lack of significance by the applied statistical test.

The ability of GSK3β to phosphorylate pathophysiologically-relevant resides of Tau is well-documented [21‐26], and Pyk2 has been shown activate GSK3β through the phosphorylation of Y216 in GSK3β’s activation loop [27‐29]. Although it has previously been demonstrated that Pyk2 can directly phosphorylate Tau at Y18 [31], whether Pyk2 can augment GSK3β’s ability to phosphorylate Tau at disease-relevant residues has not been clarified. We over-expressed combinations of GSK3β, Pyk2, Tau and Fyn in Hek293T cells and measured the phosphorylation of GSK3β at Y216 and Tau at S202/T205 (AT8) (Fig. 1A–C). Pyk2 co-transfection with GSK3β led to a significant increase in GSK3β activation, and this activation was further augmented with the addition of Fyn, a kinase known to synergistically co-activate Pyk2 [10, 45‐47] (Fig. 1A, B). Over-expression of GSK3β and Pyk2 together led to a significant increase in Tau phosphorylation at S202/T205 compared Tau co-transfected with either kinase alone (Fig. 1A, C). Over-expression of GSK3β, Pyk2 and Fyn together also led to robust Tau phosphorylation at S202/T205, however this signal failed to reach a level significantly higher than that seen with both GSK3β and Pyk2, suggesting that a ceiling to GSK3β-mediated S202/T205 phosphorylation may have been achieved in this system.

In order to confirm whether Pyk2 activity positively correlates with Tau phosphorylation at physiological levels of expression in neurons ex vivo, we conducted an acute brain slice assay in which we pharmacologically inhibited Pyk2 in mouse hippocampal slices with the selective Pyk2 inhibitor PF-719 (Fig. 2A–D). Since basal levels of AT8 Tau phosphorylation were undetectable in WT mice (Fig. 3A, G), we used tissue from transgenic PS19^0/+ mice that express a mutant form of human Tau (MAPT P301S) associated with FTD and are a well-described animal model of tauopathy [48, 49]. Unexpectedly, inhibition of Pyk2 with 1 μM PF-719 resulted in an increase of Tau phosphorylation at S202/T205 (Fig. 2A, B, D). Importantly, this increase in Tau phosphorylation occurred independently of any changes in GSK3β activity (Fig. 2A, C), suggesting that basal levels of Pyk2 activity suppress Tau phosphorylation through a GSK3β-independent mechanism.

To confirm these results in a second neuronal system, we pharmacologically inhibited Pyk2 in mature iPSC-derived human cortical neurons differentiated from iPSCs via dual SMAD inhibition and patterned into forebrain cortical neurons via Wnt inhibition. This well-described and previously validated method of obtaining cortical neurons from iPSCs has the benefit of recapitulating the differentiation of cortical neurons in the developing human brain without relying on the induced overexpression of transgenic transcription factors such as Ngn2 [40]. While Pyk2 inhibition failed to result in any measurable modulation of GSK3β activity in these neurons, the inhibition of basal Pyk2 activity with PF-719 led to significant increases in Tau phosphorylation at two pathophysiologically-relevant epitopes of Tau, S396/S404 (PHF-1) and S202/T205 (AT8) (Fig. 2E–I). Together, these results suggest that while Pyk2 can phosphorylate Tau through GSK3β when over-expressed at supra-physiological stoichiometries, basal levels of neuronal Pyk2 activity suppress Tau phosphorylation through a mechanism independent of measurable changes in GSK3β activity.

To determine whether Pyk2 expression is protective against Tau phosphorylation in an in vivo mammalian system, we again employed PS19^0/+ transgenic mice, this time interbred with a Pyk2^−/− line. To assess the influence of Pyk2 genetic deletion on Tau phosphorylation biochemically, we obtained TBS-insoluble, SDS-soluble fractions from both hippocampi and cortices of 9.5–10.5-month-old WT, Pyk2^−/−, PS19^0/+ and PS19^0/+;Pyk2^−/− animals (Fig. 3A–L). While there were no detectable levels of TBS-insoluble, SDS-soluble Tau phosphorylated at any disease-relevant epitope analyzed biochemically in WT or Pyk2^−/− mice, PS19^0/+;Pyk2^−/− animals demonstrated a significantly higher level of hippocampal Tau phosphorylation at S393/S404 (PHF-1), S262 and S199/S202 compared to PS19^0/+ mice (Fig. 3A–F). Of phospho-Tau epitopes analyzed in the cortex, only pTau T181 (AT270) demonstrated higher phosphorylation in PS19^0/+;Pyk2^−/− compared to PS19^0/+ animals, suggesting that Pyk2’s influence on regulating Tau phosphorylation may be greater in hippocampus compared to cortex (Fig. 3G–L). In support of a region-specific influence of Pyk2 in suppressing Tau phosphorylation, we observed significantly greater Pyk2 expression in the hippocampus compared to cortex both immunohistologically and biochemically (Fig. S1A–C). Pyk2 activation was also significantly higher in WT and PS19^0/+ hippocampus compared to cortex, while Tau-induced activation of Pyk2 in PS19^0/+ animals was restricted to the hippocampus (Fig. S1D). Higher levels of expression and activity of Pyk2 in the hippocampus suggest that its genetic deletion from PS19^0/+ animals would likely have a greater magnitude of effect on Tau phosphorylation in this region compared to cortex.

The ability of Pyk2 expression to suppress Tau phosphorylation, however, extends beyond hippocampus and cortex. Histological analysis of 9.5–10.5-month-old PS19^0/+ and PS19^0/+;Pyk2^−/− amygdala also revealed significant increases in immunofluorescent signal of pTau S202/T205 (AT8) in both the number of AT8-positive cell bodies, as well as the area occupied by those cell bodies, and pTau S199/S202 by mean image intensity of immunofluorescent signal (Fig. 4A–G). While sexual dimorphism with respect to severity of Tau pathology has been previously reported in PS19 transgenic mice, we observed no differences in amygdalar Tau pathology between PS19^0/+ and PS19^0/+;Pyk2^−/− animals by any measure when segregated by sex (Fig. S2) [50]. No changes were observed in total Tau immunofluorescence between PS19^0/+ and PS19^0/+;Pyk2^−/− animals, suggesting that Pyk2’s ability to suppress Tau pathology is specific to Tau phosphorylation rather than total Tau expression (Fig. S3). Taken together, these results provide both biochemical and histological evidence of Pyk2’s role in suppressing Tau phosphorylation in a well-described animal model of tauopathy in vivo.

We sought to determine whether genetic deletion of Pyk2, found here to exacerbate Tau pathology in PS19^0/+ mice, would result in appreciable changes in Tau-induced early-death or behavioral deficits in the same model. While, as expected, PS19^0/+ animals demonstrated a marked reduction in survival compared to WT and Pyk2^−/− animals, PS19^0/+;Pyk2^−/− mice showed a significant reduction in survivorship compared to PS19^0/+ animals (Fig. 5A), and segregating survivorship data by sex suggests that this effect is primarily driven by female rather than male mice (Fig. S4). Immediately preceding death, PS19^0/+ and PS19^0/+;Pyk2^−/− animals displayed a rapid, stereotyped deterioration in condition marked by hindlimb paralysis, hunched posture and reduced body weight, all of which have been previously described in this transgenic line [48]. Immunofluorescent labeling of NeuN and pTau S202/T205 (AT8) reveals substantial Tau accumulation in the lumbar enlargement of PS19^0/+ and PS19^0/+;Pyk2^−/− spinal cord, with AT8-positive immunofluorescent signal colocalizing with NeuN-positive motor neurons in the ventral horn of PS19^0/+ spinal cord (Fig. S5). In PS19^0/+;Pyk2^−/− spinal cord, AT8 immunofluorescence is considerably more diffuse, with marked loss of NeuN-positive motor neurons in the ventral horn (Fig. S5B). The presence of Tau inclusions within motor neurons of the lumbar enlargement has been previously suggested as a causal mechanism of hindlimb paralysis in PS19^0/+ mice, thus the reduced survivorship of PS19^0/+;Pyk2^−/− compared to PS19^0/+ mice, all of which succumbed to hindlimb paralysis, likely reflects an acceleration of PS19-mediated Tau pathology in these animals [48].

To assess the influence of Pyk2 expression on Tau-induced spatial memory impairment in PS19^0/+ animals, we subjected 9–10-month-old animals from all four genotypes to the Morris water maze (MWM) test. While PS19^0/+ mice of this age demonstrated no significant impairment in learning during MWM acquisition sessions, genetic deletion of Pyk2 significantly impaired MWM spatial memory acquisition in PS19^0/+;Pyk2^−/− compared to WT animals (Fig. 5B). Pyk2 deletion on the WT background did not alter performance, as reported previously [11]. To assess long-term spatial memory, animals were subjected to a probe trial 24 h after the final acquisition session. WT, Pyk2^−/− and PS19^0/+ animals all spent a significantly greater amount of time in the target quadrant compared to the opposite quadrant during the MWM probe trial. PS19^0/+;Pyk2^−/− animals, however, failed to spend significantly more time the target quadrant compared to the opposite quadrant, suggesting that Pyk2 expression is necessary to protect against Tau-induced impairment of long-term spatial memory (Fig. 5C). Impaired spatial memory observed in PS19^0/+;Pyk2^−/− mice during MWM acquisition and the probe trial could not be explained by visual impairment, as animals from all genotypes were able to effectively locate a visible platform during visual assessment (Fig. 5D). Notably, impaired spatial memory in PS19^0/+;Pyk2^−/− mice occurs prior to frank hippocampal neurodegeneration in these animals, as there were no significant reductions in hippocampal cell layer thickness across genotypes (Fig. S6).

While both PS19^0/+ and PS19^0/+;Pyk2^−/− mice weighed significantly less than WT animals, Pyk2 deletion failed to result in any measurable modulation of Tau-induced body mass reduction in PS19^0/+ mice (Fig. 5E). To assess possible Tau-induced impairments in motor coordination, grip strength and gait patterns, animals were subjected to Rotarod, wire hang and CatWalk XT assays (Fig. 5F–J). There were no observable differences across genotypes by any of these measures, suggesting that while Pyk2’s suppression of Tau pathology is concomitant with an exacerbation of Tau-induced early death and spatial memory impairment in PS19^0/+;Pyk2^−/− animals, these results are not associated with any measurable sensorimotor deficits.

To more completely elucidate the effect of genetic Pyk2 deletion on PS19^0/+ transgenic animals and to reveal the possible mechanisms by which Pyk2 suppresses Tau phosphorylation, pathology and Tau-associated phenotypes, we conducted LC-MS/MS label-free profiling of PS19^0/+ and PS19^0/+;Pyk2^−/− hippocampal tryptic peptides. Since we measured no discernable changes in either hippocampal astrogliosis or microgliosis across WT, Pyk2^−/−, PS19^0/+ and PS19^0/+;Pyk2^−/− animals (Fig. S7), we focused our analysis on neuronal, cell-autonomous mechanisms and enhanced our analysis by utilizing hippocampal synaptosomal fractions as starting material.

Proteomic analysis revealed 338 significantly differentially regulated proteins between PS19^0/+ and PS19^0/+;Pyk2^−/− hippocampal synaptosomes (Fig. 6A, B). Intriguingly, 32 (9.5%) of the identified hits were either 40S or 60S ribosomal proteins, while an additional 6 (1.8%) of identified hits were either eukaryotic translation initiation or elongation factors, all of which were upregulated in PS19^0/+;Pyk2^−/− compared to PS19^0/+ animals (Fig. 6B). Dysregulation of protein translational machinery, including altered expression of ribosomal proteins, has been observed across multiple Tau transgenic lines, and the aberrant association of Tau with ribosomal proteins, as seen in both MAPT P301L mice and AD brains, has been shown to disrupt synthesis of proteins critical for synaptic function including PSD-95 [51‐54]. Notably, the expressions of both C1qa and C1qb, two components of the classical complement system, were also significantly upregulated in PS19^0/+;Pyk2^−/− synaptosomes (Fig. 6B), suggesting that endogenous Pyk2 expression may protect against synaptic dysfunction through the suppression of synaptic C1q deposition.

Since Pyk2 is a protein kinase and Tau phosphorylation is correlated with pathophysiology, we specifically assessed phospho-enriched peptides obtained from PS19^0/+ and PS19^0/+;Pyk2^−/− hippocampal synaptosomes through LC-MS/MS label-free profiling. For each experimental replicate, relative abundance values for phospho-enriched hippocampal synaptosomal proteins were normalized to their respective total protein values to determine relative normalized phospho-protein abundance. We identified 50 significantly differentially regulated phospho-proteins between PS19^0/+ and PS19^0/+;Pyk2^−/− hippocampal synaptosomes (Fig. 6C, D). Of interest, the phosphorylation of MAPK1 (ERK2), a known regulator of Tau phosphorylation [35, 55‐57], was significantly decreased at residue Y187 in PS19^0/+;Pyk2^−/− compared to PS19^0/+ hippocampal synaptosomes. The phosphorylation of Y187 and T185, two residues present on the activation loop of MAPK1, are required for full activation of the kinase [58, 59], and thus a decrease in MAPK1 phosphorylation at Y187 suggests a decrease in MAPK1 kinase activity in PS19^0/+;Pyk2^−/− animals, which would paradoxically favor less Tau phosphorylation.

From the proteomic changes, we focused our initial attention on C1q, which is a key component of the classical complement system and, in the CNS, plays a critical role in microglia-mediated synaptic pruning during development [60]. Levels of C1q are known to increase with age, and AD brains show increased neuronal deposition of C1q in both the hippocampus and frontal cortex [61]. Expression of C1q is augmented in response to both injury and Aβ exposure, and increased levels of C1q deposition at synapses are associated with microglia-mediated synaptic engulfment and synapse loss in models of neurogenerative diseases such as AD and FTD [62, 63]. We sought to biochemically validate the proteomics-based observation of increased C1qa and C1qb expression in PS19^0/+;Pyk2^−/− hippocampal synaptosomes and assessed whether genetic Pyk2 deletion modulated Tau-induced C1q deposition in a manner that reflects Pyk2’s role in suppressing Tau-associated memory impairment in PS19^0/+ mice. We obtained synaptosomal fractions from both hippocampus (Fig. 7A–D) and cortex (Fig. 7E–H) and found that PS19^0/+;Pyk2^−/− mice displayed significantly higher levels of hippocampal, synaptosomal C1q (normalized to β-Actin) compared to WT, Pyk2^−/− and PS19^0/+ animals. In the cortex, levels of synaptosomal C1q were significantly higher in PS19^0/+;Pyk2^−/− compared to WT and PS19^0/+ mice.

Although reduced PSD-95 expression observed in PS19^0/+;Pyk2^−/− hippocampal synaptosomes only reached significance when compared to Pyk2^−/− mice (Fig. 7A, B), histological analysis revealed significant decreases in PSD-95-positive puncta in both PS19^0/+ and PS19^0/+;Pyk2^−/− dentate gyrus compared to WT and Pyk2^−/− mice (Fig. 7I, J). In the CA3 region of the hippocampus, C1q immunofluorescence signal was significantly increased in both PS19^0/+ and PS19^0/+;Pyk2^−/− animals compared to WT and Pyk2^−/− mice (Fig. 7K, L). However, in the dentate gyrus C1q immunofluorescence was significantly increased in PS19^0/+;Pyk2^−/− animals (compared to WT), while no such increase was observed in PS19^0/+ mice (Fig. 7M, N). Overall, the absence of Pyk2 enhances several aspects of complement-related synaptic damage in PS19 mice.

Our observations of exacerbated Tau pathology, early death and memory impairment in PS19^0/+;Pyk2^−/− animals suggest that Pyk2 deletion accelerates disease progression in PS19^0/+ transgenic mice. Identifying mechanisms by which Pyk2 may suppress Tau phosphorylation from proteomics-based comparison of aged-matched PS19^0/+ and PS19^0/+;Pyk2^−/− animals is therefore complicated by the fact that PS19^0/+;Pyk2^−/− animals likely represent a more advanced stage of disease. Alterations to protein translational machinery, which likely result in substantial differences in global protein translation between PS19^0/+ and PS19^0/+;Pyk2^−/− mice, further complicate the use of these animals for the identification of Pyk2-modulated regulators of Tau phosphorylation through phospho-proteomic analysis. Considering these limitations, we also conducted LC-MS/MS label-free profiling of WT and Pyk2^−/− hippocampal synaptosomal peptides, as any observed changes in the synaptic proteome between animals of these genotypes would more likely reflect the influence of Pyk2 expression per se, as opposed to differences in the stage of PS19-driven pathology.

Proteomic analysis of relative protein abundance revealed 170 significantly differentially regulated proteins between WT and Pyk2^−/− mice (Fig. 8A, B). Many of these hits represent potentially intriguing avenues for further exploration and may help elucidate Pyk2’s role in synaptic development and function [Dlg1, Dlg2, Dlg4 (PSD-95), Homer1, Camk2b, Camk2d, Nrxn1, Syt2, Gria2 (GluR2), Arhgef7 and Cask] and disease [Snca (α-synuclein) and Snca (β-synuclein)]. However, for the purposes of this paper, and in order explain Pyk2’s role in regulating signaling events that influence Tau phosphorylation, we focused our analysis on differential levels of phospho-peptides from the synaptosomal proteins with all values normalized to total protein abundance (Fig. 8C–E). We identified a total of 38 phospho-proteins that were significantly differentially regulated between WT and Pyk2^−/− animals (Fig. 8C).

In order to identify candidate regulators of Tau phosphorylation modulated by Pyk2 expression, we used STRING to generate a functional protein association network that included all 38 phospho-protein hits as well as MAPT (Tau) (Fig. 8E). Proximate regulators of Tau phosphorylation were defined a priori as kinases or phosphatases positioned one or two degrees from MAPT in the association network. Using this definition, we identified 6 possible Pyk2-modulated regulators of Tau phosphorylation (6 kinases, 0 phosphatases): Gsk3α, Gsk3β, Brsk1, Mapk1, Mark2 and Lkb1 (Stk11). Interestingly, the phosphorylation of MAPK1, the only proximate regulator of Tau phosphorylation identified in both the WT vs Pyk2^−/− and the PS19^0/+ vs PS19^0/+;Pyk2^−/− phospho-proteomic analyses, was also decreased in Pyk2^−/− hippocampal synaptosomes. However, since the activity of this kinase was decreased (rather than increased) with Pyk2 deletion, it is unlikely that this putative Tau kinase would explain Pyk2’s ability to suppress Tau phosphorylation.

To further understand the pattern of differential protein abundance and phosphorylation in hippocampal synaptosomes, we conducted Gene Ontology (GO) enrichment analysis and generated functionally-grouped network maps of significantly differentially regulated synaptic proteins identified across each proteomic and phospho-proteomic analysis. Network maps showing clusters of significantly enriched pathways (p < 0.05) were generated in ClueGO using the GO Biological Process ontology with network parameters for each analysis kept constant for proteomic (total protein) and phospho-proteomic (normalized phospho-enriched protein) hits, respectively (Fig. 9A, B, D, E). Across proteomic analyses of total hippocampal synaptosomal proteins, 25 proteins were shared between WT vs Pyk2^−/− and PS19^0/+ vs PS19^0/+;Pyk2^−/− animals, representing 14.7% of hits identified in the WT vs Pyk2^−/− analysis (Fig. 9C). GO enrichment of total proteins reveals several biological pathways conserved between WT vs Pyk2^−/− and PS19^0/+ vs PS19^0/+;Pyk2^−/− proteomic analyses, reflecting Pyk2’s role in regulating neuronal projection (GO terms: regulation of neuron projection development; regulation of plasma membrane bounded cell projection organization; regulation of plasma membrane bounded cell projection organization) and exocytosis (GO terms: regulated exocytosis; exocytosis) (Fig. 9A, B, Table 1).

Several additional pathways, however, were unique to the PS19^0/+ vs PS19^0/+;Pyk2^−/− analysis, likely reflecting Pyk2’s role in regulating aberrant, Tau-associated cellular processes specific to PS19^0/+ mice, including pathways involved in protein translation and processing (GO terms: regulation of translation; protein polymerization; negative regulation of protein metabolic process; negative regulation of cellular protein metabolic process) (Fig. 9B). Also unique to the PS19^0/+ vs PS19^0/+;Pyk2^−/− analysis are pathways involved in glial development (GO terms: glial cell development, glial cell differentiation; gliogenesis), myelination (GO term: myelination) and synaptic homeostasis (GO terms: positive regulation of synaptic transmission; regulation of synaptic plasticity; regulation of trans-synaptic signaling; chemical synaptic transmission), the latter of which may reflect impaired synaptic function in PS19^0/+;Pyk2^−/− animals. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment ontology for total protein hits identifies a single node for the PS19^0/+ vs PS19^0/+;Pyk2^−/− proteomic analysis (GO term: pathways of neurodegeneration), while no pathways were identified for the WT vs Pyk2^−/− analysis using the same parameters, suggesting that Pyk2 deletion alters pathways related to neurodegeneration in PS19^0/+, but not WT animals (Table S1).

We also generated functionally-grouped network maps for normalized phospho-enriched protein hits across phospho-proteomic analyses (Fig. 9 D, E). Encouragingly, GO enrichment analysis of WT vs Pyk2^−/− hits revealed significant enrichment for multiple biological pathways involved in protein phosphorylation (GO terms: tau-protein kinase activity; protein serine kinase activity; protein threonine kinase activity; regulation of protein phosphorylation; peptidyl-serine phosphorylation; protein phosphorylation), with “tau-protein kinase activity” having the highest percentage of associated genes of any identified pathway (11.43%) (Table 2). Using the same network parameters, we found considerably fewer enriched pathways from hits identified from the PS19^0/+ vs PS19^0/+;Pyk2^−/− analysis, with all significantly enriched pathways relating to cation homeostasis (GO terms: regulation of calcium ion transport; cellular calcium ion homeostasis; cation channel activity), likely reflecting Pyk2’s role in regulating calcium homeostasis in PS19^0/+ transgenic mice (Fig. 9E). Only 3 hits (7.9% of WT vs Pyk2^−/− hits) were shared between phospho-proteomic analyses (Fig. 9F). The lack of enriched pathways relating to protein phosphorylation in the PS19^0/+ vs PS19^0/+;Pyk2^−/− analysis further suggests that phospho-proteomic hits that may mechanistically explain Pyk2’s role in suppressing Tau phosphorylation are likely masked in PS19^0/+ animals as pathology progresses.

Because LKB1 is implicated in both Aβ processing as well as Tau phosphorylation, and thus, like Pyk2, is well-positioned to serve as possible mechanistic link between Aβ and Tau, we selected this kinase for further validation [64‐66]. To confirm whether Pyk2 modulates LKB1 (STK11) activity, we inhibited Pyk2 pharmacologically in mature iPSC-derived human cortical neurons and measured the phosphorylation of LKB1 biochemically at S428. The phosphorylation of this serine residue, located within the C-terminal domain of LKB1, is required for LKB1 nuclear export, substrate biding and activation [67‐69]. Since evidence of LKB1’s ability to directly phosphorylate Tau is lacking, we also measured the activity of p38 MAPK, a known downstream effector of LKB1 signaling [32, 33] and a direct kinase of Tau [34‐36], through the phosphorylation of T180/Y182. The phosphorylation of p38 MAPK at T180/Y182, two auto-phosphorylated residues present in the protein’s activation loop required for catalytic activity and substrate binding, are well-validated markers of p38 MAPK activity [70‐73].

In iPSC-derived human cortical neurons, Pyk2 inhibition with PF-719 significantly increased both the phosphorylation of LKB1 at S428 as well as the phosphorylation of p38 MAPK at T180/Y182, suggesting that basal levels of neuronal Pyk2 activity suppress the activation of both LKB1 and p38 MAPK in a manner that reflects Py2k’s ability to inhibit Tau phosphorylation (Fig. 10A–C). Since the phosphorylation of MAPK1 was significantly differentially regulated in both WT vs Pyk2^−/− and PS19^0/+ vs PS19^0/+;Pyk2^−/− phospho-proteomic analyses, we also assessed whether pharmacological Pyk2 inhibition would modulate MAPK1 activity via phosphorylation of its activation loop at T185/Y187 in iPSC-derived human cortical neurons. Consistent with our phospho-proteomic results suggesting decreased phosphorylation of MAPK1 in both Pyk2 and PS19^0/+;Pyk2^−/− hippocampal synaptosomes, we observed significant inhibition of MAPK1 phosphorylation at T185/Y187 with pharmacological inhibition of Pyk2, suggesting that Pyk2 positively regulates the activity of MAPK1 (Fig. S8).

We also assessed the activation of LKB1 and p38 MAPK in hippocampal tissue from 9.5–10-month old WT, Pyk2^−/−, PS19^0/+ and PS19^0/+;Pyk2^−/− animals (Fig. 10D–K). TBS-soluble LKB1 demonstrated significantly higher activation (pLKB1 S428 normalized to total LKB1) in PS19^0/+;Pyk2^−/− animals compared to WT and Pyk2^−/− mice, though no changes in TBS-soluble p38 MAPK activity (p38 MAPK T180/Y182 normalized to total p38 MAPK) were observed across genotypes (Fig. 10D–F). However, in TBS-insoluble, SDS-soluble fractions that are likely most relevant to aberrant activation and Tau accumulation, the pattern was reversed. While there were no measurable changes in the activity of SDS-soluble LKB1 (pLKB1 S428 normalized to total LKB1) across genotypes, PS19^0/+;Pyk2^−/− animals showed significantly higher activation of SDS-soluble p38 MAPK (p38 MAPK T180/Y182 normalized to total p38 MAPK) compared to WT animals (Fig. 10G–I). When normalized to β-Actin, as a measure of total available active kinase, PS19^0/+;Pyk2^−/− animals displayed increased levels of SDS-soluble p38 MAPK T182/Y182 compared to PS19^0/+ mice, though we observed no changes in levels of active, SDS-soluble LKB1 (pLKB1 S428 normalized to β-Actin) across genotypes (Fig. 10J–K).

Taken together, these results are consistent with the activities of LKB1 and p38 MAPK contributing to Pyk2’s role in protecting against Tau phosphorylation, Tau pathology, Tau-induced early death, memory impairment and C1q deposition in PS19^0/+ transgenic animals. While we provide direct evidence for endogenous Pyk2 expression restricting levels of active p38 MAPK, a known Tau kinase, in PS19^0/+ hippocampus, it remains possible that additional kinases participate in Pyk2-dependent suppression of mutant Tau phenotypes.