Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 phosphorylates GABARAPL2 at Serine 72 to enhance autophagic flux. a-f Co-IP analysis of interactions between SIK2 and LC3A ( a ), LC3B ( b ), LC3C ( c ), GABARAP ( d ), GABARAPL1 ( e ), or GABARAPL2 ( f ) in N2a cells overexpressing SIK2-flag, using anti-flag antibodies. Asterisks indicate co-precipitated ATG8 bands. Hc: IgG heavy chain. g, h Co-IP analysis of interactions between LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GABARAPL2 and SIK2 in N2a cells overexpressing respective flag-tagged ATG8 family members, using anti-flag antibodies. i, j Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with GABARAPL2 knockdown and SIK2 overexpression ( n = 3/group). k Schematic of SIK2 domain organization highlighting the kinase domain and LIR motif. Deletion mutants are illustrated below. l, m Co-IP analysis of interactions between GABARAPL2 and SIK2 deletion mutants (SIK2(1–290)-His, SIK2(1–550)-His, SIK2(1–650)-His, SIK2(1–931)-His) using anti-His antibodies. n, o Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with SIK2 deletion mutants ( n = 3/group). p Competitive binding. Purified full-length SIK2 protein were incubated with GST-GABARAPL2 and increasing concentrations of LIR peptide and subjected to GST pull-down. q Quantification of competitive binding in ( p ). r Volcano plot of differentially phosphorylated sites in N2a-APP-SIK2 versus N2a-APP cells. s, t Representative immunoblots and quantification of GABARAPL2, p62, and LC3B in N2a-APP cells with GABARAPL2 mutants ( n = 3/group). u Representative immunoblots of GABARAPL2 and p-GABARAPL2(Ser72) in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). v The quantification of p-GABARAPL2 in the dorsal hippocampus ( n = 6/group). w, x N2a-APP cells overexpressing GABARAPL2-flag were transfected with control or SIK2 for 24 h. Immunoprecipitated with anti-flag antibody and probed with anti-p-Ser72-GABARAPL2 antibody by Western blot. Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired two-tailed t -test ( x ), one-way ANOVA ( m, q ) and two-way ANOVA ( j, o, t, v ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knockdown, Over Expression, Binding Assay, Purification, Incubation, Control, Transfection, Immunoprecipitation, Two Tailed Test

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: Age-dependent downregulation of SIK2 was observed in the middle-aged AD transgenic mouse models and AD patients. a Bioinformatics analysis revealed reduced SIK2 expression in the temporal cortex of AD patients compared to controls. b Sik2 mRNA levels in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 6/group), assessed by qPCR. c Quantification of SIK2 protein levels in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 5/group), assessed by western blot. d Representative immunoblots of SIK2 in the dorsal hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice. e, f Representative immunoblots and quantification of SIK2 in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 3/group). g, h MWM performance of WT and 5 × FAD mice. Escape latency during training trials (1–7 days) ( g ). Percentage of time spent in the target quadrant during the probe trial (day 8) ( h ). i Number of platform crossings during the probe trial (day 8). j, k Linear regression analysis was performed to assess the relationship between hippocampal SIK2 protein levels and cognitive performance (percentage of time in the target quadrant ( j ) and number of platform crossings ( k )) in WT ( n = 5) and 5 × FAD mice ( n = 5). The coefficient of determination ( R 2 ) and the P value from the F-test of the overall fit are shown on the graphs. The solid line represents the line of best fit. l SIK2-specific RNA probes combined with NeuN immunofluorescence in the dentate gyrus (DG) of WT and 5 × FAD mice. Scale bar, 50 μm. m Quantification of SIK2 intensity in DG NeuN⁺ cells ( n = 3/group). n Double-label immunofluorescence showing SIK2 (red) and NeuN (green) colocalization in the DG region of WT and 5 × FAD mice. Scale bar, 50 μm. o Quantification of SIK2 intensity in DG NeuN⁺ cells ( n = 3/group). Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired Mann–Whitney test ( i ), unpaired two-tailed t -test ( a, h, m, o ), linear regression analysis ( j, k ), one-way ANOVA ( b, c, f ), and two-way ANOVA ( g ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Transgenic Assay, Expressing, Western Blot, Immunofluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 alleviates the cognitive impairment and enhances the synaptic plasticity in middle-aged 5 × FAD mice. a–e MWM performance of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Escape latency during training trials (1–7 days) ( a ) and probe trial (day 8) ( b ). Platform crossings ( c ), percentage of time in the target quadrant ( d ), and swimming speed ( e ) during the probe trial (day 8). Sample sizes: n = 21 (WT-control), n = 22 (WT-SIK2), n = 19 (5 × FAD-control), n = 19 (5 × FAD-SIK2). f, g Long-term potentiation (LTP) recordings in hippocampal CA1 regions. High-frequency stimulation (HFS) was applied, and fEPSP amplitudes were quantified during the last 10 min ( g ). Sample sizes: n = 3 mice, 7 slices per group. h Ultrastructural analysis of synapses in hippocampal CA1 region via TEM. sv, synaptic vesicle; sc, synaptic cleft; PSD, postsynaptic density. Scale bars: 1 µm (top images), 250 nm (bottom images). i, j Quantitative analysis of PSD thickness and SC width ( n = 2 mice, 5 images per mouse). k-m Dendritic morphology of CA1 pyramidal neurons. Representative images ( k ) and Sholl analysis of branch intersections ( l-m ). Scale bars, 500 µm (upper), 50 µm (middle), 10 µm (lower). Sample size: n = 7 dendrites from 3 mice per group. n, o MAP2 immunofluorescence staining in the CA1 region. Representative images ( n ) and quantitative analysis ( o ). Scale bar, 50 µm. p, q Western blot analysis of SIK2, SYN, PSD95, and BDNF in dorsal hippocampal lysates. Representative immunoblots ( p ) and quantitative analyses ( q ) ( n = 6 per group). Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b, c-g , i, j, l, m, o, q ), and three-way ANOVA ( a ) followed by the Tukey’s post-hoc test, and Scheirer-Ray-Hare test followed by the Dunn’s post-hot test ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, # P < 0.05, ## P < 0.01, & P < 0.05

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Control, Immunofluorescence, Staining, Western Blot

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 reduces Aβ deposition in 5 × FAD mice. a, b Representative immunoblots and quantitative analyses of SIK2, Aβ (6E10), APP, and BACE1 in the dorsal hippocampus of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). c, d Representative immunoblots and quantitative analyses of SIK2, Aβ (6E10), APP, and BACE1 in the dorsal hippocampus of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice ( n = 6/group). e, g Immunofluorescence staining for Aβ (6E10) in the dentate gyrus (DG) of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Representative images ( e ) and quantification ( g ) ( n = 3/group). Scale bar, 50 µm. f, h Immunofluorescence staining for Aβ (6E10) in the DG of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice. Representative images ( f ) and quantification ( h ) ( n = 3/group). Scale bar, 50 µm. i, j ELISA quantification of soluble and insoluble Aβ 1–42 levels in hippocampal homogenates of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 4/group). k, l ELISA quantification of soluble and insoluble Aβ 1–42 levels in hippocampal homogenates of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice ( n = 4/group). Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b-d , g-l ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 enhances autophagic flux in AD models. a KEGG pathway enrichment analysis showing significant activation of autophagy-related pathways in N2a-APP-SIK2 versus N2a-APP cells ( P = 0.0024, FDR < 0.05). b, c Representative immunoblots and quantitative analyses of SIK2, p62, LC3B, and APP in N2a, N2a-APP, N2a-SIK2, and N2a-APP-SIK2 cells ( n = 6). d, e Detection of lysosomal acidification in N2a, N2a-SIK2, N2a-APP, N2a-APP-SIK2, and N2a-APP-RAPA cells by mRFP-GFP-LC3 tandem fluorescence. Autophagosomes show double mRFP + GFP + signals (yellow spots), and functional autophagosomes showed mRFP + GFP - signals (red spots). Representative images ( d ) and quantization ( e ) ( n = 8–9/group). Scale bar, 10 μm. f, g Representative immunoblots and quantitative analyses of SIK2, p62, and LC3B in N2a-APP cells treated with chloroquine (CQ, 50 μmol/L, 12 h) or bafilomycin A1 (BafA1, 100 nmol/L, 12 h) ( n = 3 per group). h, i TEM images of autophagosomes (orange arrows) and autolysosomes (yellow arrows) in N2a, N2a-APP, N2a-SIK2, and N2a-APP-SIK2 cells. Scale bars, 1 µm. Quantitative analysis of autophagic vacuoles ( i ). j, k Representative immunoblots and quantitative analyses of SIK2, p62, and LC3B in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). l, m TEM images of autophagosomes (orange arrows) and autolysosomes (yellow arrows) in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Scale bars, 500 nm. Quantitative analysis of autophagic vacuoles ( m ). Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired two-way ANOVA ( c, e, g, i, k, m ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Activation Assay, Western Blot, Fluorescence, Functional Assay, Control

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 phosphorylates GABARAPL2 at Serine 72 to enhance autophagic flux. a-f Co-IP analysis of interactions between SIK2 and LC3A ( a ), LC3B ( b ), LC3C ( c ), GABARAP ( d ), GABARAPL1 ( e ), or GABARAPL2 ( f ) in N2a cells overexpressing SIK2-flag, using anti-flag antibodies. Asterisks indicate co-precipitated ATG8 bands. Hc: IgG heavy chain. g, h Co-IP analysis of interactions between LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GABARAPL2 and SIK2 in N2a cells overexpressing respective flag-tagged ATG8 family members, using anti-flag antibodies. i, j Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with GABARAPL2 knockdown and SIK2 overexpression ( n = 3/group). k Schematic of SIK2 domain organization highlighting the kinase domain and LIR motif. Deletion mutants are illustrated below. l, m Co-IP analysis of interactions between GABARAPL2 and SIK2 deletion mutants (SIK2(1–290)-His, SIK2(1–550)-His, SIK2(1–650)-His, SIK2(1–931)-His) using anti-His antibodies. n, o Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with SIK2 deletion mutants ( n = 3/group). p Competitive binding. Purified full-length SIK2 protein were incubated with GST-GABARAPL2 and increasing concentrations of LIR peptide and subjected to GST pull-down. q Quantification of competitive binding in ( p ). r Volcano plot of differentially phosphorylated sites in N2a-APP-SIK2 versus N2a-APP cells. s, t Representative immunoblots and quantification of GABARAPL2, p62, and LC3B in N2a-APP cells with GABARAPL2 mutants ( n = 3/group). u Representative immunoblots of GABARAPL2 and p-GABARAPL2(Ser72) in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). v The quantification of p-GABARAPL2 in the dorsal hippocampus ( n = 6/group). w, x N2a-APP cells overexpressing GABARAPL2-flag were transfected with control or SIK2 for 24 h. Immunoprecipitated with anti-flag antibody and probed with anti-p-Ser72-GABARAPL2 antibody by Western blot. Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired two-tailed t -test ( x ), one-way ANOVA ( m, q ) and two-way ANOVA ( j, o, t, v ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knockdown, Over Expression, Binding Assay, Purification, Incubation, Control, Transfection, Immunoprecipitation, Two Tailed Test

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: Phospho-GABARAPL2 at Ser72 alleviates the cognitive impairment of middle-aged 5 × FAD mice. a-e MWM performance of WT-control, 5 × FAD-control, 5 × FAD-PL2(WT), 5 × FAD-PL2(72A), and 5 × FAD-PL2(72E) mice. Escape latency during training trials (1–6 days) ( a ) and probe trial (day 7) ( b ). Platform crossings ( c ), percentage of time in the target quadrant ( d ), and swimming speed ( e ) during the probe trial (day 7). Sample sizes: n = 12 (WT-control), n = 10 (5 × FAD-control), n = 10 (5 × FAD-PL2(WT)), n = 10 (5 × FAD-PL2(72A)), n = 10 (5 × FAD-PL2(72E)). f, g Long-term potentiation (LTP) recordings in hippocampal CA1 regions. High-frequency stimulation (HFS) was applied, and fEPSP amplitudes were quantified during the last 10 min ( g ). Sample sizes: n = 3 mice, 7 slices per group. h, i Immunofluorescence staining for Aβ in the CA1 region. Representative images ( h ) and quantitative analysis ( i ). Scale bar, 50 µm. j, k Western blot analysis of GABARAPL2, p-GABARAPL2(Ser72), p62, LC3B, SYN, PSD95, and Aβ in dorsal hippocampal lysates. Representative immunoblots ( j ) and quantitative analyses ( k ) ( n = 6 per group). l, m TEM images of autophagic vacuoles. Representative images ( l ) and quantitative analysis ( m ). Orange arrows: double-membrane autophagosomes; yellow arrows: single-membrane autolysosomes with electron-dense contents. Scale bars, 500 nm. n KEGG pathway analysis of DEGs between 5 × FAD-control and 5 × FAD-PL2(72E) mice, showing the top 10 enriched pathways. o Proposed model illustrating how SIK2 regulates autophagosome–lysosome fusion and neurodegeneration in AD. Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b, d-g, i, k, m ), and three-way ANOVA ( a ) followed by the Tukey’s post-hoc test, and Scheirer-Ray-Hare test followed by the Dunn’s post-hot test ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Control, Immunofluorescence, Staining, Western Blot, Membrane