Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 2. Inhibition of ULK1 results in a decrease of pICln phosphorylation in vivo A, Colocalization of ULK1 and pICln phospho and total. Cells were fixed with 4% PFA and afterwards permeabilized with Triton X-100 to visualize ULK1 (red) and pICln total or phosphospecific pICln (green). The DNA was stained with DAPI (blue), scale bars 10 µm. B, HEK293T cells were treated with 30 µM ULK inhibitor MRT67307 for 5 h or transfected with 50 nM Non-targeting control pool (NT) control or ULK1 siRNA for 72 h. Cells were fixed as described in A to visualize ULK1 (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. C, HEK293T cells were treated as described in B to visualize phosphospecific pICln (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. D, Inhibition or knockdown of ULK1 decreases pICln phosphorylation in vivo. Quantification of the intensity ratio between phosphospecific pICln and pICln total was made in Fiji and the diagram in Origin, the values for MRT67307 treatment are normalized on HEK wt value, and the values for ULK1 siRNA are normalized on NT control. The significance of the values was analyzed using a Mann-Whitney U test, the p-value of HEK wt vs. MRT67307 and NT control vs. ULK1 siRNA was below 0.005, indicating significant differences between the values.

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Inhibition, Phospho-proteomics, In Vivo, Staining, Transfection, Control, Knockdown, MANN-WHITNEY

Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 4. ULK1 phosphorylation of pICln increases UsnRNP biogenesis and spliceosomal activity A, The HIV-1-based splicing reporter contains a test 5‘splice site (5‘ss) with a mutated version of viral 5′ss D4 termed „− 1G3U“. This splice site carries two nucleotide substitutions: A-to-G at position − 1 (−1 G) and A-to-U at position + 3 (+3 U) and is poorly recognized by the endogenous U1 snRNA due to a mismatch at position + 3. However, splice site recognition is efficiently restored following the co-expression of a U1 snRNA with a compensatory U-to-A nucleotide exchange at position + 6, indicating that the modified U1 is assembled into functional snRNP particles within the cell. Uppercase letters within the splice site sequences represent complementary residues, while lowercase letters represent mismatches to the U1. Mutations are highlighted in red. Base-pairing at position + 3 is highlighted by a light red box and increased font size. B; C, HEK293T cells were treated with 30 µM inhibitor MRT67307 and transiently transfected with different splicing reporter constructs (see methods section for more details). After 20 h of inhibitor treatment cells were harvested to perform RNA isolation. Quantitative RT-PCR was executed and mRNA was analyzed by assessing the respective ratio of spliced/unspliced form, the p value was below 0.005 (B). The equal averages including differences between inhibitor- treated and untreated spliced and unspliced forms are listed in C (n = 3).

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Modification, Functional Assay, Transfection, Construct, Isolation, Quantitative RT-PCR

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

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.

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

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.

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

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.

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: TUNEL Assay, Over Expression, Staining

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.

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

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 ).

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:

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.

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

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.

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

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.

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

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).

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, RNA Sequencing, Isolation, Over Expression, Electron Microscopy, Membrane

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.

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: Over Expression, Expressing, Western Blot

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.

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: RNA Sequencing, Gene Expression, Expressing

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.

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: Immunofluorescence, Staining

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 ).

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: Western Blot, Expressing, Staining, Two Tailed Test

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 ).

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

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 ).

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

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 ).

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

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots of total and phosphorylated ULK1, and total AMPKα and phospho-AMPKα Thr172 expression, in the kidneys of sham and CKD (5/6 nephrectomy) mice (n = 5). B. AMP/ATP ratio in the kidneys of WT mice in the sham and CKD groups (n = 5). C. Representative immunoblots of total AMPKα and phospho-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 5). D. AMP/ATP ratio in kidneys of WT or Ulk1 −/− mice (n = 5). E. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without 5-aminoimidazole 4-carboxamide-1-beta-d- ribofuranoside (AICAR) (1.0 mg/g BW) (n = 6). F. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P-ACC Ser79 , and P-RAPTOR Ser792 expression in liver tissue of WT or Ulk1 −/− mice treated with or without AICAR (n = 6). G. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 in white adipose tissue of WT or Ulk1 −/− mice treated with or without AICAR (n = 6). H. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without AICAR for 12 h (n = 3). P values (* P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis, Phospho-proteomics

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For ULK1 depletion experiments, Ulk1 Rat siRNA Oligo Duplex (SR514693, Origene, San Francisco, CA, USA) was used.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: Sequences of RNA and DNA oligonucleotides

Article Snippet: The sequences of short interfering (si) RNAs targeting ULK1 (si1‐ ULK1 , si2‐ ULK1 ) and the negative control (si‐NC) are listed in Table and were purchased from GenePharma Co.

Techniques: shRNA, Control, Negative Control

Journal: Stem Cells (Dayton, Ohio)

Article Title: Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1‐Dependent Autophagy

doi: 10.1002/stem.3091

Figure Lengend Snippet: PCK2 enhances osteogenic differentiation of hASCs via modulating AMPK/ULK1‐dependent autophagy. (A): Protein expression levels of p‐ULK1 (ser 555), p‐ULK1 (ser 556), p‐ULK1 (ser 757), ULK1, p‐AMPK α, and AMPK in PCK2‐knockdown and control cells are shown. (B): Western blots of p‐ULK1 (ser 555), p‐ULK1 (ser 556), p‐ULK1 (ser 757), ULK1, p‐AMPK α, and AMPK in PCK2‐expressing and control vector cells. (C, D): Relative mRNA expression levels of ULK1 (C) and RUNX2 (D) in hASCs transfected with si1‐ ULK1 , si2‐ ULK1 , and negative control (si‐NC) after 7 days of osteogenic induction. Data represent three independent experiments. (E, F): Depletion of ULK1 decreases ALP staining (E) and activity (F) on the seventh day after induction of osteogenic differentiation in hASCs. (G, H): Knockdown of ULK1 decreases ARS staining (G) and activity (H) on the 14th day after induction of osteogenic differentiation in hASCs. (I): Inhibition of AMPK in PCK2‐expressing hASCs suppresses osteogenic ability and autophagy activity of hASCs, as shown by the protein expressions of RUNX2, LC3B, and p62. (J): PCK2‐knockdown cells expressing wild‐type AMPK increase the osteogenic capacity and autophagy activity of hASCs. The protein expression patterns of RUNX2, LC3B, and p62 are presented. GAPDH was used as internal loading control in (A, B) and (I, J). ( K–N): H&E staining, Masson's trichrome staining from implanted hASC‐scaffold hybrids. Scale bar = 100 μm, n = 10. Data in this figure represent three independent experiments and are presented as mean ± SD. **, p ≤ .01; *, p ≤ .05; Student's t test. Abbreviations: ALP, alkaline phosphatase; AMPK, AMP‐activated protein kinase; ARS, alizarin red S; ATG7, autophagy‐related‐gene‐7; hASCs, human adipose‐derived stem cells; H&E, hematoxylin and eosin; LC3B, Microtubule associated protein 1 light chain 3 β; NS, not significant; OM, osteogenic media; p62, p62/SQSTM1; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2; ULK1, unc‐51 like autophagy activating kinase 1.

Article Snippet: The sequences of short interfering (si) RNAs targeting ULK1 (si1‐ ULK1 , si2‐ ULK1 ) and the negative control (si‐NC) are listed in Table and were purchased from GenePharma Co.

Techniques: Expressing, Knockdown, Control, Western Blot, Plasmid Preparation, Transfection, Negative Control, Staining, Activity Assay, Inhibition, Derivative Assay