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Santa Cruz Biotechnology shrna mediated gene silencing
Shrna Mediated Gene Silencing, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ulk1+sirna/ULK1+siRNA/pmc12803508-355-51-60
Average 94 stars, based on 36 article reviews

shrna mediated gene silencing - by Bioz Stars, 2026-09

94/100 stars

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a , Serum ULK1 (pg ml −1 ) was quantified in CU study participants at baseline ( n = 21) and 4 years later ( n = 21). n = number of study participants per experimental group. b , CSF ULK1 (pg ml −1 ) was quantified in CU controls at baseline ( n = 22) and 4 years later ( n = 22). c , Changes of hippocampal ULK1 in different age groups. Post-mortem hippocampal samples from young, middle-aged and old people ( n = 8 per group). Original WB data are provided in Extended Data Fig. . d , Tissue sections (7 µm) were prepared from HIP of human post-mortem brain tissues from middle-aged and old groups. Slides were stained for ULK1 (green), MAP2 (red) and DAPI (blue). Scale bars, 20 μm. e , f , Quantification of immunofluorescent signals in tissues ( d ) in two ways, including with the data presented as ULK1-positive (ULK1 + ) neurons/total neurons ( e ) or signal intensity per neuron ( f ) ( n = 4 and 6). g , Bar chart comparing serum ULK1 (ln transformed pg ml −1 ) in CU, AD-MCI and AD-dementia patients. Data points were subjected to ln transformation to correct for skewed data distribution. n = number of participants per group. (Age, years ± s.d., min–max: CU, 71.8 ± 6.4, 64–89 years; AD-MCI, 70.6 ± 5.4, 52–81 years; AD-dementia, 69.7 ± 6.7, 49–84 years.) h , Box plots showing changes of CSF ULK1 in designated groups. (Age, years ± s.d., min–max: CU, 72.2 ± 6.4, 64–89 years; AD-MCI, 70.7 ± 5.9, 49–81 years; AD-dementia, 70.2 ± 6.4, 49–84 years.) i , Heat map for relative abundance of ULK1 small nuclear RNA (snRNA) stratified by Braak stages (0–2, 3–4, 5–6) and brain cell types. Data represent single-nucleus RNA samples prepared from post-mortem human brain. j , k , Changes of neuronal ULK1 expression between old control and AD hippocampal tissues. ULK1 (green), MAP2 (red) and DAPI (blue). A representative set of images ( j ) and quantified data in the form of ULK1-positive (ULK1 + ) neurons/total neurons ( k ) ( n = 6 and 7). Scale bars, 20 μm ( j ). l , CDR-SB data showing that higher ULK1 correlates with slower AD progression. CSF ULK1 was measured at baseline and the patient cohort stratified into subgroups with low (50 pg ml −1 ), medium (150 pg ml −1 ) or high (250 pg ml −1 ) expression of ULK1. CDR-SB protocol was administered at 1-, 3- or 5-year follow-up assessment. The graph shows linear regression for CDR-SB score versus time. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses used were as follows: paired t -test ( a , b ), two-sided unpaired two-tailed Student’s t -test ( e , f , k ). One-way ANOVA followed by Dunnett’s multiple comparisons test ( c ) and by Tukey’s multiple comparisons test ( g, h ) and Wilcoxon test ( i ). Oli., oligodendrocytes; Ex., excitatory neurons; In., inhibitory neurons; Ast., astrocytes; Opc., oligodendrocyte progenitor cells; Mic., microglia; CI, confidence interval. * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , Serum ULK1 (pg ml −1 ) was quantified in CU study participants at baseline ( n = 21) and 4 years later ( n = 21). n = number of study participants per experimental group. b , CSF ULK1 (pg ml −1 ) was quantified in CU controls at baseline ( n = 22) and 4 years later ( n = 22). c , Changes of hippocampal ULK1 in different age groups. Post-mortem hippocampal samples from young, middle-aged and old people ( n = 8 per group). Original WB data are provided in Extended Data Fig. . d , Tissue sections (7 µm) were prepared from HIP of human post-mortem brain tissues from middle-aged and old groups. Slides were stained for ULK1 (green), MAP2 (red) and DAPI (blue). Scale bars, 20 μm. e , f , Quantification of immunofluorescent signals in tissues ( d ) in two ways, including with the data presented as ULK1-positive (ULK1 + ) neurons/total neurons ( e ) or signal intensity per neuron ( f ) ( n = 4 and 6). g , Bar chart comparing serum ULK1 (ln transformed pg ml −1 ) in CU, AD-MCI and AD-dementia patients. Data points were subjected to ln transformation to correct for skewed data distribution. n = number of participants per group. (Age, years ± s.d., min–max: CU, 71.8 ± 6.4, 64–89 years; AD-MCI, 70.6 ± 5.4, 52–81 years; AD-dementia, 69.7 ± 6.7, 49–84 years.) h , Box plots showing changes of CSF ULK1 in designated groups. (Age, years ± s.d., min–max: CU, 72.2 ± 6.4, 64–89 years; AD-MCI, 70.7 ± 5.9, 49–81 years; AD-dementia, 70.2 ± 6.4, 49–84 years.) i , Heat map for relative abundance of ULK1 small nuclear RNA (snRNA) stratified by Braak stages (0–2, 3–4, 5–6) and brain cell types. Data represent single-nucleus RNA samples prepared from post-mortem human brain. j , k , Changes of neuronal ULK1 expression between old control and AD hippocampal tissues. ULK1 (green), MAP2 (red) and DAPI (blue). A representative set of images ( j ) and quantified data in the form of ULK1-positive (ULK1 + ) neurons/total neurons ( k ) ( n = 6 and 7). Scale bars, 20 μm ( j ). l , CDR-SB data showing that higher ULK1 correlates with slower AD progression. CSF ULK1 was measured at baseline and the patient cohort stratified into subgroups with low (50 pg ml −1 ), medium (150 pg ml −1 ) or high (250 pg ml −1 ) expression of ULK1. CDR-SB protocol was administered at 1-, 3- or 5-year follow-up assessment. The graph shows linear regression for CDR-SB score versus time. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses used were as follows: paired t -test ( a , b ), two-sided unpaired two-tailed Student’s t -test ( e , f , k ). One-way ANOVA followed by Dunnett’s multiple comparisons test ( c ) and by Tukey’s multiple comparisons test ( g, h ) and Wilcoxon test ( i ). Oli., oligodendrocytes; Ex., excitatory neurons; In., inhibitory neurons; Ast., astrocytes; Opc., oligodendrocyte progenitor cells; Mic., microglia; CI, confidence interval. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The siRNA reagents used including siRNA targeting ULK1 (catalog no. SR322391, OriGene), PINK1 (catalog no. SR324912, OriGene), Parkin (catalog no. SR321228, OriGene), FUNDC1 (catalog no. SR315322, OriGene), Ambra1 (catalog no. SR310808, OriGene), BNIP3 (catalog no. SR300461, OriGene), BNIP3L/NIX (catalog no. SR300462, OriGene), GSK3-beta (catalog no. SR301979, OriGene), ULK2 (catalog no. SC-44183, Santa Cruz Biotechnology), Atg5 (catalog no. SR322789, OriGene), Beclin1 (catalog no. SR322490, OriGene), Sirt1 (catalog no. SR323581, OriGene) or scramble control siRNA Oligo Duplexes at 100 nM.

Techniques: Staining, Transformation Assay, Expressing, Control, Two Tailed Test

( a ) Western blotting showing changes of ULK1 expression in hippocampal tissues from young, middle-aged, and old humans (n = 8 per group). ( b-d ) Heat map data showing the relative abundance of ATG101 , RB1CC / FIP200 , and ATG13 mRNA stratified by Braak stages (0/2, 3/4, 5/6) and brain cell types (Oli., oligodendrocytes; Ex., excitatory neurons; In., inhibitory neurons; Ast., astrocytes; Opc., oligodendrocyte progenitor cells; Mic., microglia). Data represent single cell mRNA samples prepared from post-mortem human brain. ( e ) Changes of ULK1 signal intensity/neuron between old control and AD. ( f-k ) Changes of ULK1 in the entorhinal and prefrontal tissues between old control and AD patients. Slides were immunostained for ULK1 (green), Map2 (red), and nucleus (DAPI, blue), signals detected by immunofluorescence, and with merged images shown as indicated. Scale bars = 20 μm. Two types of data quantification were used, including data were presented as ULK1-positive (ULK1 + ) neurons/total neurons ( j , i ) or signal intensity/neuron ( h , k ). ( l ) Representative images of hippocampal brain region of AD patient of the indicated genotype stained for ULK1 (Red), Aβ plaques (6E10-positive, green) and nucleus (DAPI, blue). Scale bars = 50 μm. ( m ) Representative images of cortical brain region of AD patient of the indicated genotype stained for cTau-Lys174 (Red), ULK1 (green) and nucleus (DAPI, blue). Scale bars = 50 μm. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses were as follows: two-sided unpaired two-tailed Student’s t-test ( e , g , h , i , k ). Wilcox test ( b , c , d ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Western blotting showing changes of ULK1 expression in hippocampal tissues from young, middle-aged, and old humans (n = 8 per group). ( b-d ) Heat map data showing the relative abundance of ATG101 , RB1CC / FIP200 , and ATG13 mRNA stratified by Braak stages (0/2, 3/4, 5/6) and brain cell types (Oli., oligodendrocytes; Ex., excitatory neurons; In., inhibitory neurons; Ast., astrocytes; Opc., oligodendrocyte progenitor cells; Mic., microglia). Data represent single cell mRNA samples prepared from post-mortem human brain. ( e ) Changes of ULK1 signal intensity/neuron between old control and AD. ( f-k ) Changes of ULK1 in the entorhinal and prefrontal tissues between old control and AD patients. Slides were immunostained for ULK1 (green), Map2 (red), and nucleus (DAPI, blue), signals detected by immunofluorescence, and with merged images shown as indicated. Scale bars = 20 μm. Two types of data quantification were used, including data were presented as ULK1-positive (ULK1 + ) neurons/total neurons ( j , i ) or signal intensity/neuron ( h , k ). ( l ) Representative images of hippocampal brain region of AD patient of the indicated genotype stained for ULK1 (Red), Aβ plaques (6E10-positive, green) and nucleus (DAPI, blue). Scale bars = 50 μm. ( m ) Representative images of cortical brain region of AD patient of the indicated genotype stained for cTau-Lys174 (Red), ULK1 (green) and nucleus (DAPI, blue). Scale bars = 50 μm. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses were as follows: two-sided unpaired two-tailed Student’s t-test ( e , g , h , i , k ). Wilcox test ( b , c , d ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Western Blot, Expressing, Single Cell, Control, Immunofluorescence, Staining, Two Tailed Test

( a, b ) A schematic diagram of the strategy used to generate the Ulk1 OV mice ( a ) with PCR showing validated initial clones ( b ). ( c-e ) Representative images of primary neurons ( c ), microglia ( d ) and astrocytes ( e ) from brains of WT and Ulk1 OV mice stained for ULK1 and co-stained with Tuj1 (neuronal marker), IBA1 (microglial marker), or GFAP (a marker of astrocyte), as indicated. Scale bars = 5 μm ( c , d ) and 10 μm ( e ). ( f ) Representative western blots of ULK1 in extracts of whole brain tissue from WT and Ulk1 OV mice on postnatal day 0 (P0) (n = 3 mice/group). Actin was used as house a keeping control for quantification. ( g, h ) Representative images showing high purity of isolated primary cortical neurons ( g ) and microglia ( h ) from WT and Ulk1 OV mice. Neurons were co-stained with Tuj1 (neuronal marker) and DAPI (nucleus marker); microglia were co-stained with Iba1 (microglial marker) and DAPI. Scale bars = 100 μm. The high purity of isolated neuronal (or microglia) is indicated by the high percentage of Tuj1-positive neurons (or Iba1-positive microglia). ( i ) Western blot data of ULK1 and LC3B in whole cell extracts from HIP, PFC, and CERE of brain tissue and key organs (heart, liver, lung, kidney, muscle) from WT and Ulk1 OV mice (n = 3 female and male mice/group). HIP, hippocampus; PFC, prefrontal cortex; CERE, cerebellum. ( j ) Immunoblot analysis of different types of Aβ 1-42 prepared in this study according to in-house preparation protocol. ( k ) Representative Hoechst-staining (blue) images of cultured cortical neurons from WT and Ulk1 OV mice after 24 h exposure to the indicated reagents. Doses used were: 50 μM kainic acid (KA), 100 μM N-methyl-D-aspartate (NMDA), 5 mM 3-nitropropionic acid (3-NPA), 10 μM Rotenone, and 5 μM oAβ. Scale bars = 100 μm.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a, b ) A schematic diagram of the strategy used to generate the Ulk1 OV mice ( a ) with PCR showing validated initial clones ( b ). ( c-e ) Representative images of primary neurons ( c ), microglia ( d ) and astrocytes ( e ) from brains of WT and Ulk1 OV mice stained for ULK1 and co-stained with Tuj1 (neuronal marker), IBA1 (microglial marker), or GFAP (a marker of astrocyte), as indicated. Scale bars = 5 μm ( c , d ) and 10 μm ( e ). ( f ) Representative western blots of ULK1 in extracts of whole brain tissue from WT and Ulk1 OV mice on postnatal day 0 (P0) (n = 3 mice/group). Actin was used as house a keeping control for quantification. ( g, h ) Representative images showing high purity of isolated primary cortical neurons ( g ) and microglia ( h ) from WT and Ulk1 OV mice. Neurons were co-stained with Tuj1 (neuronal marker) and DAPI (nucleus marker); microglia were co-stained with Iba1 (microglial marker) and DAPI. Scale bars = 100 μm. The high purity of isolated neuronal (or microglia) is indicated by the high percentage of Tuj1-positive neurons (or Iba1-positive microglia). ( i ) Western blot data of ULK1 and LC3B in whole cell extracts from HIP, PFC, and CERE of brain tissue and key organs (heart, liver, lung, kidney, muscle) from WT and Ulk1 OV mice (n = 3 female and male mice/group). HIP, hippocampus; PFC, prefrontal cortex; CERE, cerebellum. ( j ) Immunoblot analysis of different types of Aβ 1-42 prepared in this study according to in-house preparation protocol. ( k ) Representative Hoechst-staining (blue) images of cultured cortical neurons from WT and Ulk1 OV mice after 24 h exposure to the indicated reagents. Doses used were: 50 μM kainic acid (KA), 100 μM N-methyl-D-aspartate (NMDA), 5 mM 3-nitropropionic acid (3-NPA), 10 μM Rotenone, and 5 μM oAβ. Scale bars = 100 μm.

Techniques: Clone Assay, Staining, Marker, Western Blot, Control, Isolation, Cell Culture

a – e , Primary cortical neurons from WT and Ulk1 OV mice were exposed to neurotoxicants for 24 h at the indicated doses, and percent neuronal death by apoptosis was quantified using a Hoechst dye: KA ( a ), NMDA ( b ), 3-NPA ( c ), rotenone ( d ), oAβ 1-42 ( e ). n = 3 biological sets. f , g , Representative images (TUNEL signal after 24 h exposure) ( f ) and quantified data (from three biological sets) ( g ). ULK1-overexpressed neurons were more resilient than WT neurons against apoptotic cell death induced by different neurotoxicants. h , i , Representative images ( h ) and a schematic diagram showing the adverse effects of Aβ and tau tangles on neuronal health ( i ). ULK1 overexpression reduced oAβ 1-42 -induced tau pathology in primary neurons. DIV 5 neurons were treated with oAβ 1-42 (5 μM) or vehicle (0.5% dimethyl sulfoxide) for 5 days and then stained with antibodies to tau (red) or MAP2 (somatodendritic compartment, green). j , k , Axonal length ( j ) and percent tau in the somatodendritic domain ( k ) in the indicated experimental groups. ULK1 overexpression reduced oAβ 1-42 -induced neuronal toxicity. One dot denotes data from one neuron ( n = 9 technical repeats from a total of 3 biological replicates). Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses were performed as follows: two-way ANOVA followed by Dunnett’s multiple comparisons test ( a – e , g ); two-way ANOVA followed by Tukey’s multiple comparisons test ( j , k ). Scale bars, 50 μm ( h ). Veh., vehicle. * P < 0.05.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a – e , Primary cortical neurons from WT and Ulk1 OV mice were exposed to neurotoxicants for 24 h at the indicated doses, and percent neuronal death by apoptosis was quantified using a Hoechst dye: KA ( a ), NMDA ( b ), 3-NPA ( c ), rotenone ( d ), oAβ 1-42 ( e ). n = 3 biological sets. f , g , Representative images (TUNEL signal after 24 h exposure) ( f ) and quantified data (from three biological sets) ( g ). ULK1-overexpressed neurons were more resilient than WT neurons against apoptotic cell death induced by different neurotoxicants. h , i , Representative images ( h ) and a schematic diagram showing the adverse effects of Aβ and tau tangles on neuronal health ( i ). ULK1 overexpression reduced oAβ 1-42 -induced tau pathology in primary neurons. DIV 5 neurons were treated with oAβ 1-42 (5 μM) or vehicle (0.5% dimethyl sulfoxide) for 5 days and then stained with antibodies to tau (red) or MAP2 (somatodendritic compartment, green). j , k , Axonal length ( j ) and percent tau in the somatodendritic domain ( k ) in the indicated experimental groups. ULK1 overexpression reduced oAβ 1-42 -induced neuronal toxicity. One dot denotes data from one neuron ( n = 9 technical repeats from a total of 3 biological replicates). Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses were performed as follows: two-way ANOVA followed by Dunnett’s multiple comparisons test ( a – e , g ); two-way ANOVA followed by Tukey’s multiple comparisons test ( j , k ). Scale bars, 50 μm ( h ). Veh., vehicle. * P < 0.05.

Techniques: TUNEL Assay, Over Expression, Staining

a , A graphic abstract depicting genotypes of parental and F1 progeny mice used in behavioral tests to assess cognitive function or dysfunction. b – d , ULK1 overexpression in the 5xFAD mice improved animals’ memory. Results of NOL ( b ), NOR ( c ) and Y-maze ( d ) assays performed in the designated four groups ( n = 31, 16, 24 and 16, respectively). Open and closed dots correspond to test results for female and male mice, respectively. e – h , ULK1 overexpression in the 5xFAD mice increased animals’ learning and memory. WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice ( n = 30, 16, 24 and 16, respectively) were subjected to MWM. Swimming trajectories were recorded during the initial 7-day training period of the MWM test ( e ). Results of probe trial test on day 8: representative images ( f ), total time in the target quadrant ( g ) and number of times mice passed the platform location ( h ). i – o , ULK1 overexpression in the 5xFAD mice reduced animals’ Aβ pathologies. Representative images of hippocampal brain region of 7.5-month-old mice stained for Aβ plaques (6E10-positive, green), a microglia marker (Iba1-positive, red) and nucleus (DAPI, blue) ( i ). The number ( j ) and size ( k ) of Aβ plaques were quantified ( n = 8, 8, 8 and 7, respectively). ELISA assays were used to detect insoluble Aβ 1-40 ( l ), insoluble Aβ 1-42 ( m ), soluble Aβ 1-40 ( n ), and soluble Aβ 1-42 ( o ) in hippocampal tissue from mice of the indicated genotypes ( n = 8 and 7). Scale bars, 1,000 μm (top rows), 100 μm (middle and bottom rows) ( i ). p – q , Total number of Iba-1 + microglia per region of interest (ROI) ( p ) and average number of microglia per Aβ plaque ( q ). Data in q from 50–132 plaques, with 7 mice per group. r , ULK1 overexpression increased microglia phagocytosis (uptake) of oAβ 1-42 ( n = 20–25 microglia per group from a total of 3 biological replicates). s , Hippocampal astrocytes (GFAP + cells per ROI) were counted in designated groups of mice ( n = 8, 8, 8 and 7 mice with a total of 23–35 randomly selected fields per group). t , Total number of Aβ plaques per ROI (6E10 + ) was determined in 2-, 7- and 19-month-old mice of the indicated genotypes. n = 5 or 8 mice per group, with 3 slides per mouse. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses were performed as follows: two-way ANOVA followed by Dunnett’s multiple comparisons test ( b – d , g , h , j , k , r – t ); repeated measures ANOVA by Turkey’s multiple comparisons test ( e ); two-sided unpaired two-tailed Student’s t -test ( l – q ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , A graphic abstract depicting genotypes of parental and F1 progeny mice used in behavioral tests to assess cognitive function or dysfunction. b – d , ULK1 overexpression in the 5xFAD mice improved animals’ memory. Results of NOL ( b ), NOR ( c ) and Y-maze ( d ) assays performed in the designated four groups ( n = 31, 16, 24 and 16, respectively). Open and closed dots correspond to test results for female and male mice, respectively. e – h , ULK1 overexpression in the 5xFAD mice increased animals’ learning and memory. WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice ( n = 30, 16, 24 and 16, respectively) were subjected to MWM. Swimming trajectories were recorded during the initial 7-day training period of the MWM test ( e ). Results of probe trial test on day 8: representative images ( f ), total time in the target quadrant ( g ) and number of times mice passed the platform location ( h ). i – o , ULK1 overexpression in the 5xFAD mice reduced animals’ Aβ pathologies. Representative images of hippocampal brain region of 7.5-month-old mice stained for Aβ plaques (6E10-positive, green), a microglia marker (Iba1-positive, red) and nucleus (DAPI, blue) ( i ). The number ( j ) and size ( k ) of Aβ plaques were quantified ( n = 8, 8, 8 and 7, respectively). ELISA assays were used to detect insoluble Aβ 1-40 ( l ), insoluble Aβ 1-42 ( m ), soluble Aβ 1-40 ( n ), and soluble Aβ 1-42 ( o ) in hippocampal tissue from mice of the indicated genotypes ( n = 8 and 7). Scale bars, 1,000 μm (top rows), 100 μm (middle and bottom rows) ( i ). p – q , Total number of Iba-1 + microglia per region of interest (ROI) ( p ) and average number of microglia per Aβ plaque ( q ). Data in q from 50–132 plaques, with 7 mice per group. r , ULK1 overexpression increased microglia phagocytosis (uptake) of oAβ 1-42 ( n = 20–25 microglia per group from a total of 3 biological replicates). s , Hippocampal astrocytes (GFAP + cells per ROI) were counted in designated groups of mice ( n = 8, 8, 8 and 7 mice with a total of 23–35 randomly selected fields per group). t , Total number of Aβ plaques per ROI (6E10 + ) was determined in 2-, 7- and 19-month-old mice of the indicated genotypes. n = 5 or 8 mice per group, with 3 slides per mouse. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses were performed as follows: two-way ANOVA followed by Dunnett’s multiple comparisons test ( b – d , g , h , j , k , r – t ); repeated measures ANOVA by Turkey’s multiple comparisons test ( e ); two-sided unpaired two-tailed Student’s t -test ( l – q ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Techniques: Over Expression, Staining, Marker, Enzyme-linked Immunosorbent Assay, Two Tailed Test

( a-e ) Behavioral assays were performed using WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Results of novel objection location (NOL) tests in female ( a , d ), male ( b , e ) or mixed-sex mice showing discrimination index ( a , b ) or % exploring frequency ( c , d , e ). Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 10 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. (f-j) Results of novel objection recognition (NOR) test. Discrimination index in female ( f ) and male ( g ) mice as well as percentage of exploring frequency showing in mixed ( h ), female ( i ), and male ( j) mice are presented. Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 11 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. ( k-m ) Distance explored by mice of different groups in the NOL ( k ), NOR ( l ), and Y maze ( m ) tests. n = 16-32 mice/group. (n, o) Spontaneous alternation was scored during the Y maze test in female (n) and male (o) mice of the indicated genotypes. Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 10 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. ( p-x ) Results of the MWM test. The numbers of mice used were n = 7-14 female mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV ; while n = 7-17 male mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV . Latency to platform during the 7-day initial training period of the MWM test ( p ); representative images of day 7 corresponding to ( p ) are shown in ( q ). Swimming speed during day 8 probe trial test using mixed sex ( r ), female ( s ) or male ( t ) mice. Time spent in the target quadrant during day 8 probe trial test using female ( u ) or male ( v ) mice. Number of times passing the platform location counted for female ( w ) or male ( x ) mice of the indicated genotypes. Open and closed dots correspond to results for female and male mice, respectively. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were Two-way ANOVA followed by Dunnett’s multiple comparisons test ( a-p , r-x ).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a-e ) Behavioral assays were performed using WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Results of novel objection location (NOL) tests in female ( a , d ), male ( b , e ) or mixed-sex mice showing discrimination index ( a , b ) or % exploring frequency ( c , d , e ). Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 10 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. (f-j) Results of novel objection recognition (NOR) test. Discrimination index in female ( f ) and male ( g ) mice as well as percentage of exploring frequency showing in mixed ( h ), female ( i ), and male ( j) mice are presented. Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 11 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. ( k-m ) Distance explored by mice of different groups in the NOL ( k ), NOR ( l ), and Y maze ( m ) tests. n = 16-32 mice/group. (n, o) Spontaneous alternation was scored during the Y maze test in female (n) and male (o) mice of the indicated genotypes. Female mice: n = 14, 7, 13 and 9 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. Male mice: n = 17, 9, 10 and 7 mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV , respectively. ( p-x ) Results of the MWM test. The numbers of mice used were n = 7-14 female mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV ; while n = 7-17 male mice for WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV . Latency to platform during the 7-day initial training period of the MWM test ( p ); representative images of day 7 corresponding to ( p ) are shown in ( q ). Swimming speed during day 8 probe trial test using mixed sex ( r ), female ( s ) or male ( t ) mice. Time spent in the target quadrant during day 8 probe trial test using female ( u ) or male ( v ) mice. Number of times passing the platform location counted for female ( w ) or male ( x ) mice of the indicated genotypes. Open and closed dots correspond to results for female and male mice, respectively. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were Two-way ANOVA followed by Dunnett’s multiple comparisons test ( a-p , r-x ).

Techniques:

( a ) Forelimb grip strength of mice with different genotypes. Open and closed dots correspond to test results for female and male mice, respectively. ( b ) Exhaustion rate of mice with different genotypes in the rotarod test. Open and closed dots correspond to test results for female and male mice, respectively. ( c-e ) Information on whole-body weight, lean tissue mass, and whole-body fat mass by low-field nuclear magnetic resonance imaging. Open and closed dots correspond to test results for female and male mice, respectively. ( f-j ) Average daily food intake, hourly oxygen consumption, hourly carbon dioxide exhalation, hourly respiratory exchange ratio (RER), and hourly energy expenditure of mice in a metabolic cage during dark/light cycles. ( k, l ) Representative images of hippocampal brain region of the indicated genotype stained for Ulk1, neuron marker (TUJ1-positive) and nucleus (DAPI, blue) ( j ). Scale bars = 50 μm. Ulk1 expression, in relative level, were quantified ( k ). n = 4 mice/group (4 slides/mouse). ( m, n ) ULK1 levels in whole brain and serum of WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice (7-month). n = 5 mice/group. ( o-q ) Western blot analysis of autophagy-related (LC3B) in hippocampal tissues from 2-and 6- month-old WT and 5xFAD mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. Representative western blot images ( o ) and quantified data in ( p, q ) are shown. n = 3 mice/group. Data are mean ± S.E.M. Statistical analyses performed were Two-way ANOVA followed by Dunnett’s multiple comparisons test ( a-j , l-n , p ). n.s., not significant.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Forelimb grip strength of mice with different genotypes. Open and closed dots correspond to test results for female and male mice, respectively. ( b ) Exhaustion rate of mice with different genotypes in the rotarod test. Open and closed dots correspond to test results for female and male mice, respectively. ( c-e ) Information on whole-body weight, lean tissue mass, and whole-body fat mass by low-field nuclear magnetic resonance imaging. Open and closed dots correspond to test results for female and male mice, respectively. ( f-j ) Average daily food intake, hourly oxygen consumption, hourly carbon dioxide exhalation, hourly respiratory exchange ratio (RER), and hourly energy expenditure of mice in a metabolic cage during dark/light cycles. ( k, l ) Representative images of hippocampal brain region of the indicated genotype stained for Ulk1, neuron marker (TUJ1-positive) and nucleus (DAPI, blue) ( j ). Scale bars = 50 μm. Ulk1 expression, in relative level, were quantified ( k ). n = 4 mice/group (4 slides/mouse). ( m, n ) ULK1 levels in whole brain and serum of WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice (7-month). n = 5 mice/group. ( o-q ) Western blot analysis of autophagy-related (LC3B) in hippocampal tissues from 2-and 6- month-old WT and 5xFAD mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. Representative western blot images ( o ) and quantified data in ( p, q ) are shown. n = 3 mice/group. Data are mean ± S.E.M. Statistical analyses performed were Two-way ANOVA followed by Dunnett’s multiple comparisons test ( a-j , l-n , p ). n.s., not significant.

Techniques: Nuclear Magnetic Resonance, Imaging, Staining, Marker, Expressing, Western Blot

(a, b) Western blot analysis of amyloid precursor protein (APP) and its cleavage fragments (APP CTFs and APP NTFs) in hippocampal tissues from WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Western blots ( a ) and quantified data ( b ) of the designated proteins in different mouse groups (n = 3 mice/group). APP-CTF – APP C-terminal fragment; APP-NTF - APP N-terminal fragment. (c-g) Representative images of hippocampal brain region in 7.5-month-old mice of the indicated genotype stained for Aβ plaques (6E10-positive, green), microglia (Iba1-positive, red), and nucleus (DAPI, blue) ( c ). Scale bars = 40 μm. Number of microglia/Aβ plaque in female ( d ) and male ( e ) mice (n = 3 or 4 mice/group, and the value from each mouse were from the data of 6 slides). Number of microglia (Iba1 + ) engulfing or near Aβ plaques in female ( f ) and male ( g ) mice. Open and closed dots correspond to data for female and male mice, respectively ( d-g ). (h) Microglia from mice of the indicated genotype were treated with Veh (DMSO) or 1uM Aβ 1-42 . Phagocytic activity of microglia was estimated based on number and proximity to Aβ plaques. Scale bars = 5 μm. (i-l) Aβ plaques and astrogliosis were quantified in hippocampal tissue from mice of the indicated genotypes. Representative images show amyloid plaques (6E10-positive, green) and astrocytes (GFAP-positive, red) in the indicated brain regions ( i ). Quantitation of astrocyte abundance based of GFAP immunofluorescence intensity in CA2-3 ( j ), CA1 ( k ) and DG ( l ) brain regions. Scale bars = 200 μm. (n = 7- 8 mice/group). (m-p) Immunofluorescence was used to quantify NeuN and DAPI staining in the hippocampal CA1 of 7-month-old mice ( m ). The thickness of neurons in hippocampal CA1 region was assessed and is shown in ( o ) (n = 4 mice per group with 8 randomly selected areas in the regions of interest (ROIs) were selected per mouse). Representative images of cortical neurons ( n ); number of neurons/ROI is shown in ( p ) (n = 4 mice per group with 4 randomly selected areas in the ROI/mouse). Scale bars = 50 μm. (q) Representative images showing hippocampal tissue from 2-, 7-, and 19-month-old mice stained for amyloid plaques (6E10-positive, green) and microglia (Iba1-positive, red). Scale bars = 1000 μm. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons ( b , j , k , l ); two-sided unpaired two-tailed Student’s t-test ( d-g )two-way ANOVA followed by Dunnett’s multiple comparisons ( o , p ). Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-sided unpaired two-tailed Student’s t-test ( d , e ); two-way ANOVA followed by Dunnett’s multiple comparisons test ( f-h , k , l , n , o , q ); repeated measures ANOVA followed by Tukey’s multiple-comparisons test ( i, j ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: (a, b) Western blot analysis of amyloid precursor protein (APP) and its cleavage fragments (APP CTFs and APP NTFs) in hippocampal tissues from WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Western blots ( a ) and quantified data ( b ) of the designated proteins in different mouse groups (n = 3 mice/group). APP-CTF – APP C-terminal fragment; APP-NTF - APP N-terminal fragment. (c-g) Representative images of hippocampal brain region in 7.5-month-old mice of the indicated genotype stained for Aβ plaques (6E10-positive, green), microglia (Iba1-positive, red), and nucleus (DAPI, blue) ( c ). Scale bars = 40 μm. Number of microglia/Aβ plaque in female ( d ) and male ( e ) mice (n = 3 or 4 mice/group, and the value from each mouse were from the data of 6 slides). Number of microglia (Iba1 + ) engulfing or near Aβ plaques in female ( f ) and male ( g ) mice. Open and closed dots correspond to data for female and male mice, respectively ( d-g ). (h) Microglia from mice of the indicated genotype were treated with Veh (DMSO) or 1uM Aβ 1-42 . Phagocytic activity of microglia was estimated based on number and proximity to Aβ plaques. Scale bars = 5 μm. (i-l) Aβ plaques and astrogliosis were quantified in hippocampal tissue from mice of the indicated genotypes. Representative images show amyloid plaques (6E10-positive, green) and astrocytes (GFAP-positive, red) in the indicated brain regions ( i ). Quantitation of astrocyte abundance based of GFAP immunofluorescence intensity in CA2-3 ( j ), CA1 ( k ) and DG ( l ) brain regions. Scale bars = 200 μm. (n = 7- 8 mice/group). (m-p) Immunofluorescence was used to quantify NeuN and DAPI staining in the hippocampal CA1 of 7-month-old mice ( m ). The thickness of neurons in hippocampal CA1 region was assessed and is shown in ( o ) (n = 4 mice per group with 8 randomly selected areas in the regions of interest (ROIs) were selected per mouse). Representative images of cortical neurons ( n ); number of neurons/ROI is shown in ( p ) (n = 4 mice per group with 4 randomly selected areas in the ROI/mouse). Scale bars = 50 μm. (q) Representative images showing hippocampal tissue from 2-, 7-, and 19-month-old mice stained for amyloid plaques (6E10-positive, green) and microglia (Iba1-positive, red). Scale bars = 1000 μm. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons ( b , j , k , l ); two-sided unpaired two-tailed Student’s t-test ( d-g )two-way ANOVA followed by Dunnett’s multiple comparisons ( o , p ). Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-sided unpaired two-tailed Student’s t-test ( d , e ); two-way ANOVA followed by Dunnett’s multiple comparisons test ( f-h , k , l , n , o , q ); repeated measures ANOVA followed by Tukey’s multiple-comparisons test ( i, j ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Western Blot, Staining, Activity Assay, Quantitation Assay, Immunofluorescence, Two Tailed Test

( a ) Schematic illustration of the workflow and data collection. Two recombinant AAV clones, AAV2/9-CMV-Ulk1-3*flag-WPRE-pA (Ulk1 AAV-OV ) and AAV2/9-U6-shRNA( Ulk-1 )-CMV-EGFP-pA, Ulk1 AAV-KD ) were individually injected bilaterally into the hippocampal region of 4.5-month-old 5×FAD mice to generate mice that overexpress or lack ULK1 in neural tissue, respectively. Equivalent viral constructs with “scrambled” target sequences were injected and the resulting mice were used as negative controls. Behavioral tests were conducted 2 months after injection. ( b ) A representative confocal image showing AAV-ULK1 (green) and NeuN (red) in neural tissue 4 weeks after AAV injection. ( c ) Quantification of co-localization of AAV-ULK1 with NeuN, and the percentage of AAV-ULK1-positive cells that were also NeuN-positive in CA regions. For each group, n = 4 mice. ( d-e ) The efficient expression of Ulk1 AAV-OV and Ulk1 AAV-KD in their respective groups experimental mice is demonstrated in a representative Western blot ( d ). Quantification of Western blot data ( e ) is shown in ( e ) (n = 3 mice/group). ( f-h ) Results of NOL ( f ), NOR ( g ) and Y-Maze ( h ) tests are presented. Tests were administered to n = 7, 8, 8, 7, 7 and 12 mice for WT(scramble), WT(Ulk1 AAV-KD ), WT(Ulk1 AAV-OV ), 5xFAD (scramble), 5xFAD (Ulk1 AAV-KD ), and 5xFAD (Ulk1 AAV-OV ) mice, respectively. Circles and squares represent the WT and 5xFAD, respectively, with open dots indicating female mice and closed dots indicating male mice. (i-m) Results of MWM assays during the initial 7-day training period are shown ( i , j ). Results of day 8 probe trial test including platform frequency ( k ) and time spent in target quadrant ( l ) are shown. A representative set of images collected during the probe trial are presented ( m ). For these experiments, n = 6, 8, 8, 7, 8 and 11 mice were used for WT(scramble), WT(Ulk1 AAV-KD ), WT(Ulk1 AAV-OV ), 5xFAD (scramble), 5xFAD (Ulk1 AAV-KD ), and 5xFAD (Ulk1 AAV-OV ), respectively. Circles and squares represent the WT and 5xFAD, respectively, with open dots indicating female mice and closed dots indicating male mice. ( n, o ) The total distance travelled (in meters) was recorded in each group of mice during the NOL ( n ) and NOR ( o ) tests. ( p ) Representative trajectories of distance traveled in the MWM test on day 7. ( q ) Data for swimming speed of each mouse group (day 8). Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-sided unpaired two-tailed Student’s t-test ( d , e ); two-way ANOVA followed by Dunnett’s multiple comparisons test ( f-h , k , l , n , o , q ); repeated measures ANOVA followed by Tukey’s multiple-comparisons test ( I , j ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Schematic illustration of the workflow and data collection. Two recombinant AAV clones, AAV2/9-CMV-Ulk1-3*flag-WPRE-pA (Ulk1 AAV-OV ) and AAV2/9-U6-shRNA( Ulk-1 )-CMV-EGFP-pA, Ulk1 AAV-KD ) were individually injected bilaterally into the hippocampal region of 4.5-month-old 5×FAD mice to generate mice that overexpress or lack ULK1 in neural tissue, respectively. Equivalent viral constructs with “scrambled” target sequences were injected and the resulting mice were used as negative controls. Behavioral tests were conducted 2 months after injection. ( b ) A representative confocal image showing AAV-ULK1 (green) and NeuN (red) in neural tissue 4 weeks after AAV injection. ( c ) Quantification of co-localization of AAV-ULK1 with NeuN, and the percentage of AAV-ULK1-positive cells that were also NeuN-positive in CA regions. For each group, n = 4 mice. ( d-e ) The efficient expression of Ulk1 AAV-OV and Ulk1 AAV-KD in their respective groups experimental mice is demonstrated in a representative Western blot ( d ). Quantification of Western blot data ( e ) is shown in ( e ) (n = 3 mice/group). ( f-h ) Results of NOL ( f ), NOR ( g ) and Y-Maze ( h ) tests are presented. Tests were administered to n = 7, 8, 8, 7, 7 and 12 mice for WT(scramble), WT(Ulk1 AAV-KD ), WT(Ulk1 AAV-OV ), 5xFAD (scramble), 5xFAD (Ulk1 AAV-KD ), and 5xFAD (Ulk1 AAV-OV ) mice, respectively. Circles and squares represent the WT and 5xFAD, respectively, with open dots indicating female mice and closed dots indicating male mice. (i-m) Results of MWM assays during the initial 7-day training period are shown ( i , j ). Results of day 8 probe trial test including platform frequency ( k ) and time spent in target quadrant ( l ) are shown. A representative set of images collected during the probe trial are presented ( m ). For these experiments, n = 6, 8, 8, 7, 8 and 11 mice were used for WT(scramble), WT(Ulk1 AAV-KD ), WT(Ulk1 AAV-OV ), 5xFAD (scramble), 5xFAD (Ulk1 AAV-KD ), and 5xFAD (Ulk1 AAV-OV ), respectively. Circles and squares represent the WT and 5xFAD, respectively, with open dots indicating female mice and closed dots indicating male mice. ( n, o ) The total distance travelled (in meters) was recorded in each group of mice during the NOL ( n ) and NOR ( o ) tests. ( p ) Representative trajectories of distance traveled in the MWM test on day 7. ( q ) Data for swimming speed of each mouse group (day 8). Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were two-sided unpaired two-tailed Student’s t-test ( d , e ); two-way ANOVA followed by Dunnett’s multiple comparisons test ( f-h , k , l , n , o , q ); repeated measures ANOVA followed by Tukey’s multiple-comparisons test ( I , j ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Recombinant, Clone Assay, shRNA, Injection, Construct, Expressing, Western Blot, Two Tailed Test

a , Representative images of Golgi staining of apical and basal dendritic spines in hippocampal CA1. b , Staining of apical dendrites in CA1 brain region of 7- to 8-month-old mice of the indicated genotype; representative images (upper) quantified data (lower). n = 4 mice per group; ≥20 dendritic fragments per mouse were counted. Scale bar, 10 μm. c , GO linked to, and hierarchical clustering (Cluster 2) of, genes differentially upregulated or downregulated in the HIP region of 5xFAD mice but normalized in 5xFAD;Ulk1 OV mice. GO biological process analysis of Cluster 2 demonstrated enrichment in pathways related to mitochondrial organization and energy production. n = 3 mice per group. RNA-seq was performed using total RNA isolated from hippocampal tissue of 7- to 8-month-old mice. d – f , ULK1 overexpression in the 5xFAD mice increased CA1 stratum pyramidale mitophagy and eliminated damaged mitochondria. Representative set of electron microscopy images ( d ), number of mitophagy events per ROI ( e ) ( n = 6 mice per group) and damaged mitochondria (percentage of total mitochondria) ( f ). For e and f , in each group a total of 39–54 random fields from 6 mice were analyzed. Scale bar, 2 μm ( d ). g , Relative hippocampal ATP levels in WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice. n = 5 mice per group. h , i , WB analysis of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in hippocampal tissues from 5xFAD and 5xFAD;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. Representative WB images ( h ) and quantified data ( i ). n = 3 mice per group. j , k , ULK1 overexpression in 5xFAD mice increased brain mitophagy as evidenced by higher colocalization of mitochondrial inner membrane protein Cox2 with the lysosomal protein cathepsin B. n = 4 mice per group. Relative colocalization of Cox2 and cathepsin B ( j ) and representative set of IF images ( k ). Scale bars, 40 μm (large panels), 2 μm (small panels) ( k ). Unless specified elsewhere, data are mean ± s.e.m. Statistical analysis used two-way ANOVA followed by Dunnett’s multiple comparisons test ( b , e – g , i , j ). BP, biological process; NADH, nicotinamide adenine dinucleotide (reduced form).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , Representative images of Golgi staining of apical and basal dendritic spines in hippocampal CA1. b , Staining of apical dendrites in CA1 brain region of 7- to 8-month-old mice of the indicated genotype; representative images (upper) quantified data (lower). n = 4 mice per group; ≥20 dendritic fragments per mouse were counted. Scale bar, 10 μm. c , GO linked to, and hierarchical clustering (Cluster 2) of, genes differentially upregulated or downregulated in the HIP region of 5xFAD mice but normalized in 5xFAD;Ulk1 OV mice. GO biological process analysis of Cluster 2 demonstrated enrichment in pathways related to mitochondrial organization and energy production. n = 3 mice per group. RNA-seq was performed using total RNA isolated from hippocampal tissue of 7- to 8-month-old mice. d – f , ULK1 overexpression in the 5xFAD mice increased CA1 stratum pyramidale mitophagy and eliminated damaged mitochondria. Representative set of electron microscopy images ( d ), number of mitophagy events per ROI ( e ) ( n = 6 mice per group) and damaged mitochondria (percentage of total mitochondria) ( f ). For e and f , in each group a total of 39–54 random fields from 6 mice were analyzed. Scale bar, 2 μm ( d ). g , Relative hippocampal ATP levels in WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice. n = 5 mice per group. h , i , WB analysis of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in hippocampal tissues from 5xFAD and 5xFAD;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. Representative WB images ( h ) and quantified data ( i ). n = 3 mice per group. j , k , ULK1 overexpression in 5xFAD mice increased brain mitophagy as evidenced by higher colocalization of mitochondrial inner membrane protein Cox2 with the lysosomal protein cathepsin B. n = 4 mice per group. Relative colocalization of Cox2 and cathepsin B ( j ) and representative set of IF images ( k ). Scale bars, 40 μm (large panels), 2 μm (small panels) ( k ). Unless specified elsewhere, data are mean ± s.e.m. Statistical analysis used two-way ANOVA followed by Dunnett’s multiple comparisons test ( b , e – g , i , j ). BP, biological process; NADH, nicotinamide adenine dinucleotide (reduced form).

Techniques: Staining, RNA Sequencing, Isolation, Over Expression, Electron Microscopy, Membrane

( a ) Representative images and quantification of basal dendritic spine density in hippocampal CA1 of WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice (n = 60 dendritic fragments from 4 mice). Scale bars = 10 μm. ( b-c ) Apical ( b ) and basal ( c ) dendritic spine density in CA3 of mice of the indicated genotypes (n = 60 dendritic fragments from 4 mice). ( d, e ) Effects of ULK1 overexpression on expression of the indicated proteins in hippocampal tissues of mice of the indicated genotypes. Representative western blots ( d ) and quantified data ( e ) (n = 3 mice/group). ( f-k ) Representative images of damaged mitochondria in entorhinal cortex (EC) ( f ) and prefrontal cortex (PFC) ( g ) brain regions from mice of the indicated genotype. Mitophagy events and percent damaged mitochondria in ( f ) and ( g ) were quantified and are shown in ( h ) to ( k ); % damaged mitochondria ( h , j ) and mitophagy ( i , k ) estimates are based on 50-200 mitochondria per group; n = 6 mice/group; approximately 20 images per/mouse. Scale bars = 1 μm. ( l ) Quantification of the indicated mRNA (n = 4 mice/group). Unless specified elsewhere, data are mean ± S.E.M. Statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons ( a-c , e , h-i ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Representative images and quantification of basal dendritic spine density in hippocampal CA1 of WT, 5xFAD, Ulk1 OV and 5xFAD;Ulk1 OV mice (n = 60 dendritic fragments from 4 mice). Scale bars = 10 μm. ( b-c ) Apical ( b ) and basal ( c ) dendritic spine density in CA3 of mice of the indicated genotypes (n = 60 dendritic fragments from 4 mice). ( d, e ) Effects of ULK1 overexpression on expression of the indicated proteins in hippocampal tissues of mice of the indicated genotypes. Representative western blots ( d ) and quantified data ( e ) (n = 3 mice/group). ( f-k ) Representative images of damaged mitochondria in entorhinal cortex (EC) ( f ) and prefrontal cortex (PFC) ( g ) brain regions from mice of the indicated genotype. Mitophagy events and percent damaged mitochondria in ( f ) and ( g ) were quantified and are shown in ( h ) to ( k ); % damaged mitochondria ( h , j ) and mitophagy ( i , k ) estimates are based on 50-200 mitochondria per group; n = 6 mice/group; approximately 20 images per/mouse. Scale bars = 1 μm. ( l ) Quantification of the indicated mRNA (n = 4 mice/group). Unless specified elsewhere, data are mean ± S.E.M. Statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons ( a-c , e , h-i ). n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Over Expression, Expressing, Western Blot

( a ) Three-dimensional (3D) principal component analysis (PCA) of RNA-seq data using hippocampal brain tissue from WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Each point represents an individual mouse (n = 3 male mice/group). ( b ) Volcano plot analysis up- or down-regulated genes in 5xFAD;Ulk1 OV and 5xFAD mice. ( c ) Heatmap showing differentially up- or down-regulated genes in WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. The color gradient from blue to white represents high to low gene expression. Hierarchical clustering identified eight gene clusters, and expression trends for each of the 8 clusters by mouse genotype are summarized to the right of the heat map. ( d ) Gene Ontology (GO) analysis of genes enriched in clusters 1-8. Different colors represent the pathways enriched in each gene cluster; some clusters show no significant enrichment.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Three-dimensional (3D) principal component analysis (PCA) of RNA-seq data using hippocampal brain tissue from WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. Each point represents an individual mouse (n = 3 male mice/group). ( b ) Volcano plot analysis up- or down-regulated genes in 5xFAD;Ulk1 OV and 5xFAD mice. ( c ) Heatmap showing differentially up- or down-regulated genes in WT, 5xFAD, Ulk1 OV , and 5xFAD;Ulk1 OV mice. The color gradient from blue to white represents high to low gene expression. Hierarchical clustering identified eight gene clusters, and expression trends for each of the 8 clusters by mouse genotype are summarized to the right of the heat map. ( d ) Gene Ontology (GO) analysis of genes enriched in clusters 1-8. Different colors represent the pathways enriched in each gene cluster; some clusters show no significant enrichment.

Techniques: RNA Sequencing, Gene Expression, Expressing

a , Breeding strategy for generating the four groups of experimental mice used in this set of experiments. b – e , Latencies to hidden platform during the 7-day training period for mice of the indicated genotypes ( b ). Representative images showing the results of the day-8 probe trial ( c ). The number of times mice passed the platform ( d ) and time spent in the target quadrant ( e ) during the probe trial. The numbers of mice used were n = 16, 11, 9 and 8 for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Open and closed dots correspond to test results for female and male mice, respectively. f , g , Representative images after immunofluorescence staining of AT8 + cells ( f ) accompanied by quantified data ( g ). For each group, n = 8 mice. Scale bars, 100 μm ( f ). h , i , The levels of p-tau and ac-tau were measured by WB from the hippocampal tissues of WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice. Representative blots ( h ) accomplished with quantifications ( i ). n = 3 mice per group. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons test ( d , e , g , i ); repeated measures ANOVA followed by Tukey’s multiple comparisons test ( b ). * P < 0.05, *** P < 0.001.

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , Breeding strategy for generating the four groups of experimental mice used in this set of experiments. b – e , Latencies to hidden platform during the 7-day training period for mice of the indicated genotypes ( b ). Representative images showing the results of the day-8 probe trial ( c ). The number of times mice passed the platform ( d ) and time spent in the target quadrant ( e ) during the probe trial. The numbers of mice used were n = 16, 11, 9 and 8 for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Open and closed dots correspond to test results for female and male mice, respectively. f , g , Representative images after immunofluorescence staining of AT8 + cells ( f ) accompanied by quantified data ( g ). For each group, n = 8 mice. Scale bars, 100 μm ( f ). h , i , The levels of p-tau and ac-tau were measured by WB from the hippocampal tissues of WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice. Representative blots ( h ) accomplished with quantifications ( i ). n = 3 mice per group. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons test ( d , e , g , i ); repeated measures ANOVA followed by Tukey’s multiple comparisons test ( b ). * P < 0.05, *** P < 0.001.

Techniques: Immunofluorescence, Staining

( a, b ) Number of platform location crossings for female ( a ) and male ( b ) mice of different genotypes in the MWM test trial. Data were shown as mean ± S.E.M. (Female mice: n = 10, 7,4 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Male mice: n = 6, 4, 5 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively). ( c, d ) Time spent in the target quadrant for female ( c ) and male mice ( d ) from different genotypes in the MWM. Data are shown as mean ± S.E.M. (Female mice: n = 10, 7,4 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Male mice: n = 6, 4, 5 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively). ( e, f ) The levels of p-Tau and ac-Tau were measured by Western blot of hippocampal tissues of WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice. A set of representative blots ( e ) accomplished with quantifications ( f ) are shown. n = 3 mice/group. ( g ) Western blot data showing the expression of Sirt1 and ac-p53 in hippocampal tissues from designated mouse groups. (n = 3 mice/group). ( h ) Representative images with immunofluorescent staining of acTau-Lys174 (red) and aggregated Tau puncta (green) in HEK293 Tau-Venus cells. ( I, j ) Representative images with immunofluorescent staining of AT8 (p-Tau Ser202 and Thr205) and IBA1(microglia) in hippocampi of hTau.P301S and hTau.P301S;Ulk1 OV mice with quantification of the numbers of microglia presented (n = 6 mice/group). Unless specified elsewhere, data are mean ± S.E.M. Statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons ( a-d , f ); two-sided unpaired two-tailed Student’s t-test ( g ).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a, b ) Number of platform location crossings for female ( a ) and male ( b ) mice of different genotypes in the MWM test trial. Data were shown as mean ± S.E.M. (Female mice: n = 10, 7,4 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Male mice: n = 6, 4, 5 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively). ( c, d ) Time spent in the target quadrant for female ( c ) and male mice ( d ) from different genotypes in the MWM. Data are shown as mean ± S.E.M. (Female mice: n = 10, 7,4 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively. Male mice: n = 6, 4, 5 and 4 mice for WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV , respectively). ( e, f ) The levels of p-Tau and ac-Tau were measured by Western blot of hippocampal tissues of WT, hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice. A set of representative blots ( e ) accomplished with quantifications ( f ) are shown. n = 3 mice/group. ( g ) Western blot data showing the expression of Sirt1 and ac-p53 in hippocampal tissues from designated mouse groups. (n = 3 mice/group). ( h ) Representative images with immunofluorescent staining of acTau-Lys174 (red) and aggregated Tau puncta (green) in HEK293 Tau-Venus cells. ( I, j ) Representative images with immunofluorescent staining of AT8 (p-Tau Ser202 and Thr205) and IBA1(microglia) in hippocampi of hTau.P301S and hTau.P301S;Ulk1 OV mice with quantification of the numbers of microglia presented (n = 6 mice/group). Unless specified elsewhere, data are mean ± S.E.M. Statistical significance was determined using two-way ANOVA followed by Dunnett’s multiple comparisons ( a-d , f ); two-sided unpaired two-tailed Student’s t-test ( g ).

Techniques: Western Blot, Expressing, Staining, Two Tailed Test

a , b , Representative WB images ( a ) and quantified data ( b ) of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in hippocampal tissues from hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. n = 3 mice per group. c , d , Representative WB images ( c ) and quantified data ( d ) of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in primary cortical neurons from hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor bafilomycin A1 (Baf1) to assess autophagic flux. n = 3 biological sets. e , NAD + levels in the hippocampal tissue of four groups of mice ( n = 4 mice per group). f , Changes of Sirt1 gene expression in designated groups ( n = 4 mice per group). g , h , Quantification of Sirt1 ( g ) and ac-p53 ( h ) protein levels in designated groups ( n = 3 mice per group). Original WB data are shown in Extended Data Fig. . i , j , Overexpression of ULK1 inhibits acTau-lys174 and tau tangles. Quantified data from acTau-lys174 ( i ) and tau aggregation ( j ) in HEK293 cells expressing 0N4R P301S Tau-Venus ( n = 14 technical repeats from a total of 3 biological replicates). k , l , Representative images ( k ) and quantification data ( l ) of ac-Tau174 staining of primary cortical neurons from hTau.P301S and hTau.P301S;Ulk1 OV mice treated with or without the SIRT1 inhibitor selisistat. Nuclei were stained with DAPI. n = 12 technical repeats from a total of 3 biological replicates. Scale bars, 50 μm ( k ). m , Schematic diagram of the proposed pathway. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons test ( b , d – j , l ).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , b , Representative WB images ( a ) and quantified data ( b ) of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in hippocampal tissues from hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor leupeptin to assess autophagic flux. n = 3 mice per group. c , d , Representative WB images ( c ) and quantified data ( d ) of autophagy-related (LC3B) and mitophagy-related proteins (mitochondrial inner membrane protein MT-CO2/Cox2 and the lysosomal protein cathepsin B) in primary cortical neurons from hTau.P301S, Ulk1 OV and hTau.P301S;Ulk1 OV mice treated with the autophagy‑lysosome inhibitor bafilomycin A1 (Baf1) to assess autophagic flux. n = 3 biological sets. e , NAD + levels in the hippocampal tissue of four groups of mice ( n = 4 mice per group). f , Changes of Sirt1 gene expression in designated groups ( n = 4 mice per group). g , h , Quantification of Sirt1 ( g ) and ac-p53 ( h ) protein levels in designated groups ( n = 3 mice per group). Original WB data are shown in Extended Data Fig. . i , j , Overexpression of ULK1 inhibits acTau-lys174 and tau tangles. Quantified data from acTau-lys174 ( i ) and tau aggregation ( j ) in HEK293 cells expressing 0N4R P301S Tau-Venus ( n = 14 technical repeats from a total of 3 biological replicates). k , l , Representative images ( k ) and quantification data ( l ) of ac-Tau174 staining of primary cortical neurons from hTau.P301S and hTau.P301S;Ulk1 OV mice treated with or without the SIRT1 inhibitor selisistat. Nuclei were stained with DAPI. n = 12 technical repeats from a total of 3 biological replicates. Scale bars, 50 μm ( k ). m , Schematic diagram of the proposed pathway. Unless specified elsewhere, data are mean ± s.e.m. Statistical analyses performed were two-way ANOVA followed by Dunnett’s multiple comparisons test ( b , d – j , l ).

Techniques: Membrane, Gene Expression, Over Expression, Expressing, Staining

a , Tau puncta formation (left) and degradation of preformed tau puncta (right) were quantified in HEK293 cells expressing 0N4R P301S Tau-Venus in the presence or absence of ULK1 activator Rac-(BL)-918. n = a total of 50 randomly selected fields per group from 3 biological replicates. b , c , HEK293 P301S tau-Venus cells were transfected with siRNA-ULK1 ( b ), siRNA-ULK2 ( c ) or siRNA-scramble ( b , c ), and transfected cells were incubated with or without 5-μM Rac-(BL)-918 in the presence of 10-nM tau seeds. Graphs in a – c show normalized fluorescence intensity relative to vehicle control ( a ) or in the absence of Rac-(BL)-918 ( b , c ), as indicated. n = a total of 54 randomly selected fields per group from 3 biological replicates. d , Rac-(BL)-918 inhibited the formation of tau puncta dependent on ULK1, FUNDC1, PINK1, Ambra1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918 simultaneously. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. e , Rac-(BL)-918 increased the degradation of preformed tau puncta dependent on ULK1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by subsequent treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. f , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the presence of siRNA targeting unc-51/ULK1 or control siRNA in the presence of Rac-(BL)-918, where indicated. Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 3 biological sets. g , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the absence or presence of siRNA to pink-1 or fndc-1/FUNDC1 . Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 5 biological sets. In the box plots, the center line denotes the median, box range indicates the 25th–75th percentile and whiskers denote minimum and maximum values. Unless specified elsewhere, data are mean ± s.e.m. Results represent pooled data from three to five biological replicates. Violin and box plots are centered around the median with interquartile ranges and all data points shown; the shape of the plot reflects the distribution of data. Statistical significance was determined using the Kruskal–Wallis test (non-normal data distribution) or one-way ANOVA (normal data distribution) ( a – e ) or two-way ANOVA followed by Tukey’s multiple comparisons test ( f , g ).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: a , Tau puncta formation (left) and degradation of preformed tau puncta (right) were quantified in HEK293 cells expressing 0N4R P301S Tau-Venus in the presence or absence of ULK1 activator Rac-(BL)-918. n = a total of 50 randomly selected fields per group from 3 biological replicates. b , c , HEK293 P301S tau-Venus cells were transfected with siRNA-ULK1 ( b ), siRNA-ULK2 ( c ) or siRNA-scramble ( b , c ), and transfected cells were incubated with or without 5-μM Rac-(BL)-918 in the presence of 10-nM tau seeds. Graphs in a – c show normalized fluorescence intensity relative to vehicle control ( a ) or in the absence of Rac-(BL)-918 ( b , c ), as indicated. n = a total of 54 randomly selected fields per group from 3 biological replicates. d , Rac-(BL)-918 inhibited the formation of tau puncta dependent on ULK1, FUNDC1, PINK1, Ambra1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918 simultaneously. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. e , Rac-(BL)-918 increased the degradation of preformed tau puncta dependent on ULK1 and Beclin1. HEK293 P301S tau-Venus cells were transfected with siRNA-scramble or a siRNA candidate, followed by subsequent treatment with 10-nM tau seeds and 5-μM Rac-(BL)-918. n = a total of 36–40 randomly selected fields per group from 3 biological replicates. f , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the presence of siRNA targeting unc-51/ULK1 or control siRNA in the presence of Rac-(BL)-918, where indicated. Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 3 biological sets. g , Associative memory tests were administered to adult day-2 transgenic C. elegans expressing hTau[P301L] n -sid-1 OV in the absence or presence of siRNA to pink-1 or fndc-1/FUNDC1 . Data are shown in the format of % CI, where a lower score corresponds to greater response to odorant and better memory function. n = 5 biological sets. In the box plots, the center line denotes the median, box range indicates the 25th–75th percentile and whiskers denote minimum and maximum values. Unless specified elsewhere, data are mean ± s.e.m. Results represent pooled data from three to five biological replicates. Violin and box plots are centered around the median with interquartile ranges and all data points shown; the shape of the plot reflects the distribution of data. Statistical significance was determined using the Kruskal–Wallis test (non-normal data distribution) or one-way ANOVA (normal data distribution) ( a – e ) or two-way ANOVA followed by Tukey’s multiple comparisons test ( f , g ).

Techniques: Expressing, Transfection, Incubation, Fluorescence, Control, Transgenic Assay

( a ) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on Tau seed-induced formation of Tau puncta in HEK293 cells expressing 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. (b) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on degradation of preformed Tau puncta in the HEK293 cells expression 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. ( c ) mt-Keima and YPH-Parkin expressing HeLa cells were used to evaluate the effects on mitophagy induction by treating a positive control CCCP. Representative images are shown. ( d-f ) Quantification of mitophagy events by treating ULK1 activators, Rac-BL-918 (0.5, 5 µM) and LYN-1604 (2, 4 µM), as well as ULK1 inhibitors SBI-0206965 (5, 10 µM) and XST-14 (2.5, 5 µM). One dot represents the average value of one image. Data were pooled from 3 biological replicates. ( g-h ) Representative images ( g ) and quantification ( h ) of mitophagy induction from primary cortical neurons by treating ULK1 activators, Rac-BL-918 (5 µM) and LYN-1604 (4 µM), as well as ULK1 inhibitors SBI-0206965 (5 µM) and XST-14 (5 µM). Mitophagy was detected using a mitophagy detection dye kit. Nuclei were stained with DAPI. Scale bar = 20 μm. ( i ) Representative blots of target proteins (ULK1, ULK2, PINK1, GSK3β, Parkin, Atg5, FUNDC1, AMBRA1, BNIP3, Nix, Beclin1) and loading control (GAPDH or β-tubulin) in HEK293 cells expressing 0N4R P301S Tau-Venus transfected with siRNAs (100 nM, 48 h) or scrambled siRNA. ( j ) Associative memory tests were administered to adult day 2 transgenic C. elegans expressing hTau[P301L]n-sid-1 OV in the presence of ULK1 inhibitors (10, 100 µM of SBI-0206965; 5, 50 µM of XST-14). Attraction to odorant is quantified as Chemotaxis Index (% CI), where lower score corresponds to greater response to odorant/better memory function. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were one-way ANOVA followed by Dunnett’s multiple comparisons test ( a , b , d-f , h ); two-way ANOVA followed by Tukey’s multiple-comparisons test ( j ).

Journal: Nature Aging

Article Title: Reduced ULK1 links impaired autophagy and mitophagy to Alzheimer’s disease pathology

doi: 10.1038/s43587-026-01108-z

Figure Lengend Snippet: ( a ) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on Tau seed-induced formation of Tau puncta in HEK293 cells expressing 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. (b) Effects of ULK1 activators (2, 4 µM of LYN-1604 dihydrochloride; 0.5, 5 µM of (Rac)-BL-918) and inhibitors (5, 10 µM of SBI-0206965; 2.5, 5 µM of XST-14) on degradation of preformed Tau puncta in the HEK293 cells expression 0N4R P301S Tau-Venus. Data are pooled from 3 biological replicates. ( c ) mt-Keima and YPH-Parkin expressing HeLa cells were used to evaluate the effects on mitophagy induction by treating a positive control CCCP. Representative images are shown. ( d-f ) Quantification of mitophagy events by treating ULK1 activators, Rac-BL-918 (0.5, 5 µM) and LYN-1604 (2, 4 µM), as well as ULK1 inhibitors SBI-0206965 (5, 10 µM) and XST-14 (2.5, 5 µM). One dot represents the average value of one image. Data were pooled from 3 biological replicates. ( g-h ) Representative images ( g ) and quantification ( h ) of mitophagy induction from primary cortical neurons by treating ULK1 activators, Rac-BL-918 (5 µM) and LYN-1604 (4 µM), as well as ULK1 inhibitors SBI-0206965 (5 µM) and XST-14 (5 µM). Mitophagy was detected using a mitophagy detection dye kit. Nuclei were stained with DAPI. Scale bar = 20 μm. ( i ) Representative blots of target proteins (ULK1, ULK2, PINK1, GSK3β, Parkin, Atg5, FUNDC1, AMBRA1, BNIP3, Nix, Beclin1) and loading control (GAPDH or β-tubulin) in HEK293 cells expressing 0N4R P301S Tau-Venus transfected with siRNAs (100 nM, 48 h) or scrambled siRNA. ( j ) Associative memory tests were administered to adult day 2 transgenic C. elegans expressing hTau[P301L]n-sid-1 OV in the presence of ULK1 inhibitors (10, 100 µM of SBI-0206965; 5, 50 µM of XST-14). Attraction to odorant is quantified as Chemotaxis Index (% CI), where lower score corresponds to greater response to odorant/better memory function. Unless specified elsewhere, data are mean ± S.E.M. Statistical analyses performed were one-way ANOVA followed by Dunnett’s multiple comparisons test ( a , b , d-f , h ); two-way ANOVA followed by Tukey’s multiple-comparisons test ( j ).

Techniques: Expressing, Positive Control, Staining, Control, Transfection, Transgenic Assay, Chemotaxis Assay

Integrated analyses identify and validate α‐Amyrin as a DLK‐binding compound. (a) Analysis of binding between αA (1 µ m ) and 468 commercially available kinases; red circles identify potential binding interactions, and circle size corresponds to binding affinity. (b) 3D representation of induced fit docking of αA and DLK based on prior structure analysis of DLK; DLK shown in gray, binding domains in green, and αA in blue. (c–e) Representative Western blot results of thermal shift assay (c), and quantification of αA‐induced shift in DLK thermal stability (d) and actin thermal stability (e). (f) Co‐localization of SARM1 and ULK1 in HEK293 P301S Tau‐Venus cells in the absence or presence of Tau seeds (10 n m ) with or without αA (1 µ m , 72 h). (g) Reciprocal co‐IP of SARM1 and ULK1 in HEK293 P301S Tau‐Venus cells with or without αA (1 µM, 72 h) treatment after Tau seeding. Input and IgG controls are shown. (h) Western blot of the indicated proteins in HEK293 P301S Tau‐Venus cells treated with vehicle or αA (1 µ m , 72 h). (i) Tau puncta formation in HEK293 P301S Tau‐Venus cells in the presence or absence of DLK inhibitor GNE3511, SARM1 inhibitor DSRM3716, or ULK1 inhibitor MRT68921 treatment with αA (1 µ m , 72 h). (j) Tau puncta formation in HEK293 P301S Tau‐Venus cells after SiRNA knock down of DLK or ULK1 genes. Cells were treated with vehicle or αA (1 µ m , 72 h) as indicated. Unless specified elsewhere, data are mean ± S.E.M from 3 biological repeats. Statistical analyses used were as follows: Repeated measures ANOVA by Tukey's multiple‐comparisons test (d, e); one‐way ANOVA followed by Dunnett's multiple comparisons test (f); one‐way ANOVA followed by Tukey's multiple‐comparisons test (i, j). All panels: n.s ., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: Advanced Science

Article Title: The Mitochondrial Guardian α‐Amyrin Mitigates Alzheimer's Disease Pathology via Modulation of the DLK‐SARM1‐ULK1 Axis

doi: 10.1002/advs.202512374

Figure Lengend Snippet: Integrated analyses identify and validate α‐Amyrin as a DLK‐binding compound. (a) Analysis of binding between αA (1 µ m ) and 468 commercially available kinases; red circles identify potential binding interactions, and circle size corresponds to binding affinity. (b) 3D representation of induced fit docking of αA and DLK based on prior structure analysis of DLK; DLK shown in gray, binding domains in green, and αA in blue. (c–e) Representative Western blot results of thermal shift assay (c), and quantification of αA‐induced shift in DLK thermal stability (d) and actin thermal stability (e). (f) Co‐localization of SARM1 and ULK1 in HEK293 P301S Tau‐Venus cells in the absence or presence of Tau seeds (10 n m ) with or without αA (1 µ m , 72 h). (g) Reciprocal co‐IP of SARM1 and ULK1 in HEK293 P301S Tau‐Venus cells with or without αA (1 µM, 72 h) treatment after Tau seeding. Input and IgG controls are shown. (h) Western blot of the indicated proteins in HEK293 P301S Tau‐Venus cells treated with vehicle or αA (1 µ m , 72 h). (i) Tau puncta formation in HEK293 P301S Tau‐Venus cells in the presence or absence of DLK inhibitor GNE3511, SARM1 inhibitor DSRM3716, or ULK1 inhibitor MRT68921 treatment with αA (1 µ m , 72 h). (j) Tau puncta formation in HEK293 P301S Tau‐Venus cells after SiRNA knock down of DLK or ULK1 genes. Cells were treated with vehicle or αA (1 µ m , 72 h) as indicated. Unless specified elsewhere, data are mean ± S.E.M from 3 biological repeats. Statistical analyses used were as follows: Repeated measures ANOVA by Tukey's multiple‐comparisons test (d, e); one‐way ANOVA followed by Dunnett's multiple comparisons test (f); one‐way ANOVA followed by Tukey's multiple‐comparisons test (i, j). All panels: n.s ., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ULK1 (Human) – 3 unique 27mer siRNA duplexes (SR322391), MAP3K12 (Human) – 3 unique 27mer siRNA duplexes (SR305269) were purchased from OriGene.

Techniques: Binding Assay, Western Blot, Thermal Shift Assay, Co-Immunoprecipitation Assay, Knockdown

( A ) Schematic of primary and validation screens. Readout is based on quantification of red mKeima puncta per cell. Representative images of siControl and siULK1 demonstrate image segmentation and feature collection of perinuclear mKeima puncta from acid pH (excitation ~586 nm). ( B ) Screening results plotted as log 2 of the mean FC of the three siRNAs for each gene relative to the negative control against the −log 10 of the combined P value. Selected candidates (RSA analysis) are annotated in red. ( C ) Venn diagram depicts overlap of significant hits from both cell lines of the validation screen with the FC criteria <0.5. ( D ) Lysosomal mKeima puncta for 19 candidates fulfilling the selection criteria from (C). Data are depicted as the mean of two siRNAs from two independent repetitions. Data normalized to positive (ULK1) and negative (Control) control siRNAs. ( E and F ) Representative images (60×) of Tig3 cells (72-hour transfection) in full medium (FM) or starved in HBSS for 2 hours, immunostained for LC3B (E) or ATG16L1 (F). Nuclear stain with Hoechst 33342. Magnified inserts of highlighted areas on the right. Scale bar, 5 μm. The number of puncta was quantified from ≥100 cells, and the data represent mean ± SD number of puncta per cell. ATG16L1 ( n = 2) and LC3B ( n = 3). **** P < 0.0001. ( G ) TEM analysis of Tig3 cells (72-hour transfection) starved for 2 hours (HBSS) in the presence of 200 nM BafA1 before fixation. Representative images are shown. Scale bar, 1 μm. Images displayed on the right are cropped and magnified from the highlighted area. Red arrows denote autophagic vesicles. Quantification of mean number of autophagic vesicles ± SD per cross section ( n ≥ 25 cross sections). Statistical analysis [(E) to (G)] was performed by unpaired Student’s t test. ** P < 0.01, **** P < 0.0001.

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A ) Schematic of primary and validation screens. Readout is based on quantification of red mKeima puncta per cell. Representative images of siControl and siULK1 demonstrate image segmentation and feature collection of perinuclear mKeima puncta from acid pH (excitation ~586 nm). ( B ) Screening results plotted as log 2 of the mean FC of the three siRNAs for each gene relative to the negative control against the −log 10 of the combined P value. Selected candidates (RSA analysis) are annotated in red. ( C ) Venn diagram depicts overlap of significant hits from both cell lines of the validation screen with the FC criteria <0.5. ( D ) Lysosomal mKeima puncta for 19 candidates fulfilling the selection criteria from (C). Data are depicted as the mean of two siRNAs from two independent repetitions. Data normalized to positive (ULK1) and negative (Control) control siRNAs. ( E and F ) Representative images (60×) of Tig3 cells (72-hour transfection) in full medium (FM) or starved in HBSS for 2 hours, immunostained for LC3B (E) or ATG16L1 (F). Nuclear stain with Hoechst 33342. Magnified inserts of highlighted areas on the right. Scale bar, 5 μm. The number of puncta was quantified from ≥100 cells, and the data represent mean ± SD number of puncta per cell. ATG16L1 ( n = 2) and LC3B ( n = 3). **** P < 0.0001. ( G ) TEM analysis of Tig3 cells (72-hour transfection) starved for 2 hours (HBSS) in the presence of 200 nM BafA1 before fixation. Representative images are shown. Scale bar, 1 μm. Images displayed on the right are cropped and magnified from the highlighted area. Red arrows denote autophagic vesicles. Quantification of mean number of autophagic vesicles ± SD per cross section ( n ≥ 25 cross sections). Statistical analysis [(E) to (G)] was performed by unpaired Student’s t test. ** P < 0.01, **** P < 0.0001.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Negative Control, Selection, Control, Transfection, Staining

( A and C ) Tig3 cells (72-hour transfection) were starved for indicated intervals in HBSS (A) or starved for 16 hours before treatment with cycloheximide (CHX) (100 ng/ml) for indicated intervals (C). A representative Western blot is shown ( n = 3). ( B and D ) Densiometric quantifications relating to (A) and (C). Data represent relative ULK1 expression normalized to time point zero and total loading and are depicted as the mean ± SEM from three independent experiments. Statistical analysis was performed by two-way ANOVA. * P < 0.05 (B) and P = 0.0685 (D). ( E ) Western blot of liver tissue lysates from starved WT and KO mice. A representative blot from three mice of each genotype is shown. [(A), (C), and (E)] Gel stain is used as a loading control. ( F ) Densiometric quantification relating to (E). The graph depicts relative expression of ULK1 normalized to total loading. The data represent mean ± SD ( n = 6). Statistical analysis was performed by unpaired Student’s t test. ** P < 0.01. ( G ) Representative images from immunohistochemistry of starved mouse livers stained for ULK1. Scale bar 100 μm. ( H ) Representative images (60×) of WT and Peli3 KO cells starved for 2 hours in HBSS and immunostained for ULK1. Nuclear stain with Hoechst 33342. Magnified inserts shown on the right. Scale bar, 5 μm. ( I ) Quantifications relating to (H). ULK1 puncta quantified from ≥120 cells. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed by unpaired Student’s t test. **** P < 0.0001. ( J ) Representative Western blot of primary hepatocytes from Peli3 WT and KO mice. Starvation and BafA1 treatments were for 4 hours ( n = 3). ( K ) Densiometric quantification of phospho-ATG14 (P-ATG14) (Ser 29 ) and total ATG14 (T-ATG14) relating to (J). Data depict the mean ± SD from four independent experiments. Statistical analyses by one-way ANOVA. * P < 0.05.

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A and C ) Tig3 cells (72-hour transfection) were starved for indicated intervals in HBSS (A) or starved for 16 hours before treatment with cycloheximide (CHX) (100 ng/ml) for indicated intervals (C). A representative Western blot is shown ( n = 3). ( B and D ) Densiometric quantifications relating to (A) and (C). Data represent relative ULK1 expression normalized to time point zero and total loading and are depicted as the mean ± SEM from three independent experiments. Statistical analysis was performed by two-way ANOVA. * P < 0.05 (B) and P = 0.0685 (D). ( E ) Western blot of liver tissue lysates from starved WT and KO mice. A representative blot from three mice of each genotype is shown. [(A), (C), and (E)] Gel stain is used as a loading control. ( F ) Densiometric quantification relating to (E). The graph depicts relative expression of ULK1 normalized to total loading. The data represent mean ± SD ( n = 6). Statistical analysis was performed by unpaired Student’s t test. ** P < 0.01. ( G ) Representative images from immunohistochemistry of starved mouse livers stained for ULK1. Scale bar 100 μm. ( H ) Representative images (60×) of WT and Peli3 KO cells starved for 2 hours in HBSS and immunostained for ULK1. Nuclear stain with Hoechst 33342. Magnified inserts shown on the right. Scale bar, 5 μm. ( I ) Quantifications relating to (H). ULK1 puncta quantified from ≥120 cells. Data represent the mean ± SD from three independent experiments. Statistical analysis was performed by unpaired Student’s t test. **** P < 0.0001. ( J ) Representative Western blot of primary hepatocytes from Peli3 WT and KO mice. Starvation and BafA1 treatments were for 4 hours ( n = 3). ( K ) Densiometric quantification of phospho-ATG14 (P-ATG14) (Ser 29 ) and total ATG14 (T-ATG14) relating to (J). Data depict the mean ± SD from four independent experiments. Statistical analyses by one-way ANOVA. * P < 0.05.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Transfection, Western Blot, Expressing, Staining, Control, Immunohistochemistry

$( A ) Co-IP from Tig3 cells expressing GFP or GFP-PELI3 in full medium or starved (HBSS, 2 hours). Western blot of inputs and IP fractions are shown from a representative experiment ( n = 3). ( B and C ) Top: Schematic of ULK1 (B) or PELI3 (C) domain organization and relevant deletion constructs. Bottom: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( D ) Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). L.E, low exposure; H.E, high exposure. ( E ) Left: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. ** P < 0.01. ( F ) Co-IP from HEK293T cells expressing indicated constructs and treated with BafA1 or MG132 as indicated (2 hours). Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( G ) Co-IP of endogenous ULK1 from starved WT and Peli3 KO mouse liver tissue. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( H ) Left: Co-IP of endogenous ULK1 from WT and Peli3 KO mouse-derived primary hepatocytes in full medium or starved. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait from starved samples. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. * P < 0.05.$

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: ( A ) Co-IP from Tig3 cells expressing GFP or GFP-PELI3 in full medium or starved (HBSS, 2 hours). Western blot of inputs and IP fractions are shown from a representative experiment ( n = 3). ( B and C ) Top: Schematic of ULK1 (B) or PELI3 (C) domain organization and relevant deletion constructs. Bottom: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( D ) Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). L.E, low exposure; H.E, high exposure. ( E ) Left: Co-IP from HEK293T cells expressing indicated constructs. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. ** P < 0.01. ( F ) Co-IP from HEK293T cells expressing indicated constructs and treated with BafA1 or MG132 as indicated (2 hours). Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( G ) Co-IP of endogenous ULK1 from starved WT and Peli3 KO mouse liver tissue. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). ( H ) Left: Co-IP of endogenous ULK1 from WT and Peli3 KO mouse-derived primary hepatocytes in full medium or starved. Western blot analysis of inputs and IP fractions are shown from a representative experiment ( n = 3). Right: Corresponding densiometric quantification of ubiquitin levels normalized to bait from starved samples. The data represent mean ± SD ( n = 3). Statistical analysis performed by unpaired Student’s t test. * P < 0.05.

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, Construct, Derivative Assay

In response to nutrient deprivation, PELI3 safeguards ULK1 homeostasis through ubiquitination of ULK1. This is facilitated by PELI3’s interaction with ATG8 proteins. In the absence of PELI3, ULK1 accumulates, leading to a disruption of autophagy and a decreased degradation of LDs in the liver, causing enhanced hepatic steatosis. Created with BioRender (Frankel, 2023; https://BioRender.com/a71f180 ).

Journal: Science Advances

Article Title: Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis

doi: 10.1126/sciadv.adr2450

Figure Lengend Snippet: In response to nutrient deprivation, PELI3 safeguards ULK1 homeostasis through ubiquitination of ULK1. This is facilitated by PELI3’s interaction with ATG8 proteins. In the absence of PELI3, ULK1 accumulates, leading to a disruption of autophagy and a decreased degradation of LDs in the liver, causing enhanced hepatic steatosis. Created with BioRender (Frankel, 2023; https://BioRender.com/a71f180 ).

Article Snippet: For screen validations, PELI3 siRNAs (4392420, 4392420), Control siRNA (4390844), and ULK1 siRNA (s15963) were purchased from Thermo Fisher Scientific.

Techniques: Disruption

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