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Genetic Ablation of Hematopoietic Cell Kinase Accelerates Alzheimer’s Disease–Like Neuropathology in Tg2576 Mice

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Abstract

Microglial dysregulation, pertaining to impairment in phagocytosis, clearance and containment of amyloid-β (Aβ), and activation of neuroinflammation, has been posited to contribute to the pathogenesis of Alzheimer’s disease (AD). Detailed cellular mechanisms that are disrupted during the disease course to display such impairment in microglia, however, remain largely undetermined. We hypothesize that loss of hematopoietic cell kinase (HCK), a phagocytosis-regulating member of the Src family tyrosine kinases that mediate signals from triggering receptor expressed on myeloid cells 2 and other immunoreceptors, impairs microglial homeostasis and Aβ clearance, leading to the accelerated buildup of Aβ pathology and cognitive decline during the early stage of neuropathological development. To elucidate the pivotal role of HCK in AD, we generated a constitutive knockout of HCK in the Tg2576 mouse model of AD. We found that HCK deficiency accelerated cognitive decline along with elevated Aβ level and plaque burden, attenuated microglial Aβ phagocytosis, induced iNOS expression in microglial clusters, and reduced pre-synaptic protein at the hippocampal regions. Our findings substantiate that HCK plays a prominent role in regulating microglial neuroprotective functions and attenuating early AD neuropathology.

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Acknowledgments

We thank the following people for their kind contribution and assistance: Dr. Clifford Lowell (University of California, San Francisco) for the HCK-KO mice; Dr. David H. Cribbs (University of California, Irvine) for the Tg2576 mice; Dr. David Baglietto-Vargas (University of California, Irvine) for his technical support on Confocal Microscopy; and Dr. Samuel E. Marsh (University of California, Irvine) for his guidance on Imaris analysis.

Authorships

All protein extraction, MSD, western blot analysis, and statistical analyses were conducted by SLL. Guidance on microdissection and immunostaining were provided by JZ. Animal husbandry and genotyping were done by SLL, ZK, EV, and CC, and tissue collection was conducted by SLL, ZK, JZ, and CJRO. Behavioral tests were designed by CJRO and executed by CJRO and SLL. NOR and OIP scoring was done by SLL and CC. Tissue sectioning was done by ZK, CC, and SG, while immunostaining, imaging, and analyses were done by SLL, DNT, CC, and VG. Experiments were conceived and designed by MK and SLL. Manuscript was written by SLL and critically reviewed by MK. All authors read and approved the final manuscript.

Funding

This study was supported by the National Institute of Health/National Institute of Environmental Health Sciences (R01 ES024331) and Alzheimer’s Association (NIRG-12-242598 and AARF-16-440554).

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Authors and Affiliations

  1. Department of Medicine, Center for Occupational and Environmental Health, University of California, Irvine, CA, 92617, USA

    Siok Lam Lim, Joannee Zumkehr, Carlos J. Rodriguez-Ortiz & Masashi Kitazawa

  2. Molecular and Cell Biology, University of California, Merced, CA, 95343, USA

    Siok Lam Lim, Diana Nguyen Tran, Zanett Kieu, Christine Chen, Emmanuel Villanueva, Sagar Ghiaar, Victoria Gallup, Joannee Zumkehr, Carlos J. Rodriguez-Ortiz & Masashi Kitazawa

  3. Department of Neurology, University of California, Irvine, CA, 92697, USA

    David H. Cribbs

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  1. Siok Lam Lim
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Correspondence to Masashi Kitazawa.

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Fig. S1 Knocking out HCK in Tg2576 mice did not modulate the recognition index (RI) of NOR and OIP nor the motor skills in MWM test. (a-d) WT, HCK-KO, Tg2576 and Tg/HCK-KO mice of 10-12 months were subjected to a battery of behavioral tests, namely NOR (a), OIP (b), and MWM (c-d). There were no significant differences in the RI of NOR (a) and OIP (b) tests between the genotypes during the 1, 3 and 5 mins of the experiments. Data are expressed as mean ± SEM with n = 7-22. All mouse genotypes completed the 1 wk MWM test with no apparent differences in the total distance moved (c) or in the swim speed (d). Data are expressed as mean ± SEM with n = 9-13

Fig. S2 Knocking out HCK in Tg2576 mice moderately modulated the intensity of PSD95 in mouse hippocampus.(a) Representative images of PSD95 (post-synaptic protein marker) at the DG, CA1 and CA3 regions of the hippocampus of WT, HCK-KO, Tg2576 and Tg/HCK-KO mice (11-13 months old). Scale bar, 50 µm. (b) Quantitative analyses of % PSD95 intensities in WT, HCK-KO, Tg2576 and Tg/HCK-KO mice taken relative to that of HCK-KO mice revealed lowest, but not significant, protein expression in Tg/HCK-KO mice among all genotypes. Data are expressed as mean ± SEM from three sections per mouse with n = 7-9. (c) Representative immunoblots of synaptophysin and PSD95 were shown. Tubulin was probed as loading control. (d) Quantitative analysis of synaptophysin and PSD95 band intensities after normalized to that of tubulin did not show significant differences between genotypes. Data are expressed as mean ± SEM with n = 5-8 per genotype

Fig. S3 HCK deficiency in Tg2576 mice did not modulate APP production and processing as well as BACE1 activity. (a) Representative immunoblots of full length and CTFs APP expression in hippocampal lysates of WT, HCK-KO, Tg2576 and Tg/HCK-KO mice using 6E10 and CT20 antibodies, respectively. GAPDH was probed as protein loading control. (b) Quantitative analysis of full length (6E10) and CTFs: C83 and C99 (CT20) band intensities after normalized to that of GAPDH. Deleting HCK in Tg2576 mice did not modulate the expression of full length APP nor that of fragments C83 and C99 from that of Tg2576 mice. However, they were highly expressed in the AD mice than in the WT or HCK-KO mice as expected. Data are expressed as mean ± SEM with n = 6-8 per genotype. ** p < 0.01, *** p < 0.001 and **** p < 0.0001 between indicated genotypes. (c) Representative immunoblots of immature (70 kDa) and mature (80 kDa) BACE1 expression in hippocampal lysates of WT, HCK-KO, Tg2576 and Tg/HCK-KO mice. Tubulin was probed as protein loading control. (d) Quantitative analysis of immature and mature BACE1 band intensities after normalized to that of tubulin. There were no apparent differences between the genotypes in both forms of BACE1. Data are expressed as mean ± SEM with n = 5-8 per genotype

Fig. S4 HCK deletion in Tg2576 mice resulted in apparent reduction of Thioflavin-S sphericity and Iba1+ microglia processes length. (a) Representative confocal images of Thioflavin-S plaque (green) clustered by Iba1+ (red) microglia in Tg2576 (left) and Tg/HCK-KO (right) mice (11-13 months old). Scale bar, 20 µm. Knocking out HCK reduced the sphericity/compactness of Thioflavin-S plaques by 24% when compared to Tg2576 mice (b). Quantification of Thioflavin-S plaque volume covered by Iba1+ cells per plaque volume per cell (c) did not show significant differences between the two mouse genotypes. Neither did volumetric and Imaris automated analyses of Iba1+ cells clustering around Thioflavin-S plaques showed significant differences in the number of cells per plaque volume (d), nor % Iba1+ cells clustering Thioflavin-S plaques per plaque volume per cell (e). Nonetheless, there was near significant reduction in the total processes length of Iba1+ microglia per microglia (f), but no drastic alteration in the microglia branching in Tg/HCK-KO mice (g). Plaques were analyzed in the hemibrains (excluding the thalamus) of 7 Tg2576 (n = 76) and 7-9 Tg/HCK-KO mice (n = 80-92). Data are expressed as mean ± SEM from one section per mouse

Fig. S5 HCK deficiency in Tg2576 mice significantly reduced the number of CD11b+ cells in microglial clusters. Clusters of microglial cells positively stained for CD11b revealed significant difference in the number of CD11b+ cells between Tg2576 and Tg/HCK-KO mice. Microglial clusters were analyzed in the hemibrains (excluding the thalamus) of 7 Tg2576 (n = 34) and 11 Tg/HCK-KO mice (n = 67). Data are expressed as mean ± SEM from 1 section per mouse. ** p < 0.01 relative to Tg2576 mice

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Lim, S.L., Tran, D.N., Kieu, Z. et al. Genetic Ablation of Hematopoietic Cell Kinase Accelerates Alzheimer’s Disease–Like Neuropathology in Tg2576 Mice. Mol Neurobiol 57, 2447–2460 (2020). https://doi.org/10.1007/s12035-020-01894-6

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  • DOI: https://doi.org/10.1007/s12035-020-01894-6

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