TY - JOUR
T1 - RanBP9 aggravates synaptic damage in the mouse brain and is inversely correlated to spinophilin levels in Alzheimer's brain synaptosomes
AU - Palavicini, J. P.
AU - Wang, H.
AU - Bianchi, E.
AU - Xu, S.
AU - Rao, J. S.
AU - Kang, D. E.
AU - Lakshmana, M. K.
N1 - Funding Information:
Acknowledgements. This work was supported by National Institute of Aging (NIA)/NIH grant numbers (1R03AG032064-01, M.K. Lakshmana), 1R01AG036859-01, M.K. Lakshmana). We are grateful to receive Alzheimer’s and control brain tissues provided by the ‘Harvard Brain Tissue Resource Center, which is supported in part by PHS grant number R24MH068855′ at the McLean Hospital.
Funding Information:
euthanized under isoflurane anesthesia and frontal cortices from WT, APDE9 and APDE9/RanBP9 mice were weighed and dounced in a grinder using Syn-PER synaptic protein extraction reagent (cat# 87793) purchased from Thermo Scientific (Rockford, IL, USA). Alzheimer’s and control brain tissues were provided by the ‘Harvard Brain Tissue Resource Center’, which is supported in part by PHS grant number R24MH068855′′ at the McLean hospital. Immediately before use, protease inhibitor mixture for mammalian cells from Sigma (cat# P8340, St. Louis, MO, USA) was added to the Syn-PER reagent. Human brain tissue was similarly dounced using Syn-PER synaptic protein extraction reagent at volume ratio of ten using a 7-ml Dounce tissue grinder with 15 up and down even strokes. The homogenate was centrifuged at 2000 g for 10 min to remove cell debris. The resulting supernatant was centrifuged at 15 000 g for 20 min. The supernatant formed the cytosolic fraction and the synaptosome pellet was gently resuspended in Syn-PER synaptic protein extraction reagent. The amount of total proteins in the homogenate, cytosolic fraction and synaptosomes were measured by BCA method and compared. The quality of synaptosome preparation was verified by immunoblotting for two cytosolic proteins (TGFb and stathmin), two nuclear proteins (PCNA and HDAC2) and two synaptic proteins (synaptophysin and PSD-95).
PY - 2013/6
Y1 - 2013/6
N2 - We previously demonstrated that overexpression of RanBP9 led to enhanced Aβ generation in a variety of cell lines and primary neuronal cultures, and subsequently, we confirmed increased amyloid plaque burden in a mouse model of Alzheimer's disease (AD). In the present study, we found striking reduction of spinophilin protein levels when RanBP9 is overexpressed. At 12 months of age, we found spinophilin levels reduced by 70% (P<0.001) in the cortex of APΔE9/RanBP9 mice compared with that in wild-type (WT) controls. In the hippocampus, the spinophilin levels were reduced by 45% (P<0.01) in the APΔE9/RanBP9 mice. Spinophilin immunoreactivity was also reduced by 22% (P<0.01) and 12% (P<0.05) in the cortex of APΔE9/RanBP9 and APΔE9 mice, respectively. In the hippocampus, the reductions were 27% (P<0.001) and 14% (P<0.001) in the APΔE9/RanBP9 and APΔE9 mice, respectively. However, in the cerebellum, spinophilin levels were not altered in either APΔE9 or APΔE9/RanBP9 mice. Additionally, synaptosomal functional integrity was reduced under basal conditions by 39% (P<0.001) in the APΔE9/RanBP9 mice and ∼23% (P<0.001) in the APΔE9 mice compared with that in WT controls. Under ATP-and KCl-stimulated conditions, we observed higher mitochondrial activity in the WT and APΔE9 mice, but lower in the APΔE9/RanBP9 mice. Significantly, we confirmed the inverse relationship between RanBP9-N60 and spinophilin in the synaptosomes of Alzheimer's brains. More importantly, both APΔE9 and APΔE9/RanBP9 mice showed impaired learning and memory skills compared to WT controls. These data suggest that RanBP9 might play a crucial role in the loss of spines and synapses in AD.
AB - We previously demonstrated that overexpression of RanBP9 led to enhanced Aβ generation in a variety of cell lines and primary neuronal cultures, and subsequently, we confirmed increased amyloid plaque burden in a mouse model of Alzheimer's disease (AD). In the present study, we found striking reduction of spinophilin protein levels when RanBP9 is overexpressed. At 12 months of age, we found spinophilin levels reduced by 70% (P<0.001) in the cortex of APΔE9/RanBP9 mice compared with that in wild-type (WT) controls. In the hippocampus, the spinophilin levels were reduced by 45% (P<0.01) in the APΔE9/RanBP9 mice. Spinophilin immunoreactivity was also reduced by 22% (P<0.01) and 12% (P<0.05) in the cortex of APΔE9/RanBP9 and APΔE9 mice, respectively. In the hippocampus, the reductions were 27% (P<0.001) and 14% (P<0.001) in the APΔE9/RanBP9 and APΔE9 mice, respectively. However, in the cerebellum, spinophilin levels were not altered in either APΔE9 or APΔE9/RanBP9 mice. Additionally, synaptosomal functional integrity was reduced under basal conditions by 39% (P<0.001) in the APΔE9/RanBP9 mice and ∼23% (P<0.001) in the APΔE9 mice compared with that in WT controls. Under ATP-and KCl-stimulated conditions, we observed higher mitochondrial activity in the WT and APΔE9 mice, but lower in the APΔE9/RanBP9 mice. Significantly, we confirmed the inverse relationship between RanBP9-N60 and spinophilin in the synaptosomes of Alzheimer's brains. More importantly, both APΔE9 and APΔE9/RanBP9 mice showed impaired learning and memory skills compared to WT controls. These data suggest that RanBP9 might play a crucial role in the loss of spines and synapses in AD.
KW - RanBP9
KW - mitochondrial activity
KW - spines
KW - spinophilin
KW - synaptosomes
KW - transgenic mice
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U2 - 10.1038/cddis.2013.183
DO - 10.1038/cddis.2013.183
M3 - Article
C2 - 23764848
AN - SCOPUS:84879637595
SN - 2041-4889
VL - 4
JO - Cell Death and Disease
JF - Cell Death and Disease
IS - 6
M1 - e667
ER -