Protective Effect Of Ndfip1 On Aβ Oligomers Induced Neurotoxic Injury

Objectives:Alzheimer’s disease（AD）is a degenerative disease of the central nervous system.Its main characteristics are persistent cognitive decline and brain function decline.However,the precise mechanisms underlying AD pathogenesis remain unresolved.Abnormal metabolism of iron,copper,zinc and other metal ions may contribute to the pathology of Alzheimer’s disease（AD）,and involved in the secretion and deposition ofβ-Amyloid（Aβ）.Aβis a small-molecule hydrolyzed peptide of amyloid precursor protein（APP）,which aggregates from Aβoligomers and fibrillar Aβin extracellular space.fibrillar Aβconstitute the core component of senile plaques,and Aβoligomers are more toxicity than fibrillar Aβ.Aβoligomers may be a key molecule that causes neuronal damage.Therefore,the expression and regulation of proteins related to metal ion metabolism are considered to be an important part of maintaining ion homeostasis.Divalent metal transporter 1（DMT1）is responsible for the uptake of a broad range of divalent metal ions,including iron ion.our research group have found that DMT1 is abnormal distribution and expression in AD patients and animal models,which may also aggravate Aβoligomers neurotoxicity.Nedd4 family interacting protein 1（Ndfip1）was discovered in 2000.it can specifically bind to the Nedd4 protein family,which promote the ubiquitination and degradation of DMT1,inhibit the transport function of DMT1,and reduce the level of iron ions in the cytoplasm of neurons.Ndfip1 was reported to protect neurons against Fe²⁺-induced neurotoxicity,be responsible for neuron survival after brain trauma,antagonize 6-hydroxydopamine（6-OHDA）-induced dopaminergic neuron damage under the synergistic effect of iron and 1-methyl-4-phenylpyridine（MPP⁺）-Induced Neuronal apoptosis.APP/PS1 transgenic mice undergo pathological changes in the brain that are highly similar to those in clinical cases,and hence are effective tools for studying AD.In our previous experiments,Ndfip1 expression in the cortex and hippocampus was lower in APP/PS1 mice than in wild-type（WT）mice.Immunofluorescence double-labeling with Ndfip1 and Aβshowed that Ndfip1 was co-localized with Aβin senile plaques in APP/PS1 mouse brain.Based on these findings,we speculate that Ndfip1downregulation promotes AD development.As the abnormal distribution and low expression of Ndfip1,the ubiquitination process was inhibited,which can lead to the accumulation and functional abnormality of related Target proteins and cause neuronal death.Ndfip1 might be a potential molecular target for the AD prevention and treatment.To analyze the significant role of Ndfip1in the pathogenesis of AD,we constructed transgenic mice that specifically overexpress Ndfip1 in the brain.Behavioral,morphological,and molecular biology analyses of these mice were performed to uncover the molecular mechanisms whereby Ndfip1 antagonizes Aβ_1-42oligomers（AβOs）-induced neuronal toxicity,as well as a theoretical basis for AD treatment.Methods:Construction and characterization of transgenic mice with Ndfip1 overexpression in the brain（termed Ndfip1 Tg mice）1.Using the Piggy BAC transposase system and fertilized egg injection,iZEG-NDFIP1 was randomly inserted into the genome of a C57BL/6 mouse to obtain the first Ndfip1-overexpressing mouse.2.iZEG-NDFIP1 transgenic-positive mice and Camk IIa-Cre transgenic mice were interbred.The tail DNA of the offspring was analyzed via polymerase chain reaction（PCR）and Lac Z staining.3.Overexpression of Ndfip1 in the cortex and hippocampus was confirmed in4-month-old Ndfip1 Tg mice via immunofluorescence staining,confocal laser scanning,and western blotting.The expression of the Ndfip1 gene was assessed via reverse transcription PCR.4.Physiological indicators such as body weight were monitored in Ndfip1 Tg mice at various ages.5.Nesting experiments were performed to evaluate the daily behavior of Ndfip1 Tg mice.6.The iron content in the cortex,hippocampus,substantia nigra,striatum,liver,spleen,kidneys,and blood of 4-month-old transgenic mice was determined using atomic absorption spectroscopy（AAS）.Evaluation of the effects of Ndfip1 overexpression on AβOs-induced neurotoxicity1.Four-month-old Ndfip1 Tg mice and WT C57BL/6 mice were reared in separate cages,each with 2 or 3 mice.Both sets of mice were divided into a control group（Ndfip1 Tg,WT）and an AβOs group（Ndfip1 Tg/AβOs,WT/AβOs）with 6–8 mice per group.Biological indicators such as body weight were measured before and after AβOs injection.2.Neuronal damage model was prepared by injecting soluble AβOs into WT mice and Ndfip1 Tg mice.5μl of normal saline（control groups）or an AβOs solution（AβOs groups）was slowly injected into the lateral ventricle.3.The Morris water maze test was used to evaluate spatial cognition and learning and memory abilities.4.After behavioral assessment,a morphological analysis of the brain tissue was performed.The distribution and expression of Neu N,neuronal damage in the cortex and hippocampus,and neuronal apoptosis in the cortex and hippocampus were assessed via immunohistochemical,Nissl,and terminal deoxynucleotidyl transferase d UTP nick end labeling（TUNEL）staining,respectively.5.Levels of postsynaptic compact protein 95（PSD95）and synapse protein 1（SYN1）in the cortex and hippocampus were monitored via western blotting and their corresponding m RNAs via real-time PCR.Protection mechanism of Ndfip1 overexpression in the brain on AβOs-induced neuronal toxicity1.The distribution and expression of cleaved（active）caspase-3 and divalent transporter protein 1（DMT1）were determined via immunohistochemistry.2.Western blotting was used to detect the following proteins in the cortex and hippocampus:caspase-3,cleaved caspase-3,ERK1/2,phosphorylated（active）ERK1/2（p-ERK1/2）,Ndfip1,and DMT1.3.AAS was used to measure iron content in the cortex and hippocampus.4.Reactive oxygen species（ROS）in the cortex and hippocampus were quantified using an ROS kit.5.The concentrations of 3-neurotrophin,4-hydroxynonenal,and 8-oxo-2’-deoxyguanosine in the cortex and hippocampus were measured using an enzyme-linked immunosorbent assay kit.Results:1.A line of transgenic mice stably overexpressing Ndfip1 in the brain was generated through breeding and genotyping.2.Ndfip1 expression in the cytoplasm of neurons in the cerebral cortex and hippocampus was higher in Ndfip1 Tg mice than in WT mice（P<0.01）.3.Body weight measured 1,4,8,and 12 months after birth was similar in age-matched Ndfip1 Tg and WT mice.The ratio of organ weight to body weight and nesting behavior did not differ significantly in 4-month-old Ndfip1 Tg and WT mice.4.The iron content in the cortex,hippocampus,substantia nigra,striatum,liver,kidney,spleen,and blood did not differ significantly between Ndfip1 Tg and WT mice.5.In the Morris water maze test,the average escape latency and search distance tended to decrease over time in all four mouse groups（WT,Ndfip1 Tg,WT/AβOs,Ndfip1 Tg/AβOs）.Both parameters were significantly higher in the WT/AβOs versus the WT group（P<0.05）and significantly lower in the Ndfip1 Tg/AβOs versus the WT/AβOs group（P<0.05）.WT,Ndfip1 Tg and Ndfip1 Tg/AβOs mice stayed somewhat in the target area and entered the target area significantly more often than did WT/AβOs mice（P<0.05）.6.Immunohistochemical staining for Neu N in the brains of WT and Ndfip1 Tg mice showed no obvious neuronal damage.The cortex and hippocampal vertebral neurons were arranged neatly,and the morphology was complete.WT/AβOs mice had fewer Neu N-positive neurons in the cortex and hippocampus than did WT mice（P<0.01）,and the vertebral neurons in the hippocampus area were disordered.Ndfip1 Tg/AβOs mice had more Neu N-positive neurons than did WT/AβOs mice（P<0.01）.7.The nerve cells in the cortex and hippocampus CA1 area of WT and Ndfip1 Tg mice were densely packed and neatly arranged.The cytoplasm was rich in Nissl bodies,which were blue to dark purple;the nuclei were light blue.WT/AβOs mice had neuronal edema and fewer cells and cytoplasmic Nissl bodies than did WT mice（P<0.01）;the cells were sparsely arranged and had large intracellular spaces,while the Nissl bodies had unclear boundaries and stained lavender.The number of cells and Nissl bodies was significantly higher in Ndfip1 Tg/AβOs versus WT/AβOs mice（P<0.01）.8.There were almost no TUNEL-positive cells in the brain in either WT or Ndfip1 Tg mice.WT and Ndfip1 Tg mice in the AβOs group had significantly more TUNEL-positive neurons in the cortex and hippocampus area than did their counterparts in the control group（P<0.01）.Ndfip1 Tg/AβOs mice had significantly fewer apoptotic cells in two areas than did WT/AβOs mice（P<0.01）.9.Western blotting showed significantly reduced expression and activity of PSD95and SYN1 in the cortex and hippocampus of WT/AβOs versus WT mice（P<0.05）.The levels of these proteins were higher in Ndfip1 Tg/AβOs mice than in WT/AβOs mice（P<0.05）.m RNA levels（determined via real-time PCR）corresponded with protein levels in all four mouse groups.10.Ndfip1 levels in the cortex and hippocampus were significantly lower in WT/AβOs mice than in WT and Ndfip1 Tg/AβOs mice on western blots（P<0.05）.Western blotting and immunohistochemistry showed increased and more widespread expression of DMT1 in the cortex and hippocampus of WT/AβOs mice（P<0.05）and reduced expression of DMTI in Ndfip1 Tg/AβOs versus WT/AβOs mice（P<0.05）.11.ROS levels were higher in WT/AβOs mice than in WT mice（P<0.05）and lower in Ndfip1 Tg/AβOs mice than in WT/AβOs mice（P<0.05）.12.Caspase-3 activity was higher in the AβOs groups than in the control groups（P<0.05）;this difference was significant for WT mice（P<0.05）but not for Ndfip1 Tg mice.On immunohistochemistry,the number of cells expressing cleaved caspase-3 in the cortex and hippocampus area was significantly higher in the WT/AβOs group versus the WT group（P<0.05）and significantly lower in the Ndfip1 Tg/AβOs group versus the WT/AβOs group（P<0.05）.13.As shown via western blotting,ERK1/2 levels in the cortex and hippocampus were similar in all four groups.The ratio of p-ERK1/2 to ERK1/2 was significantly higher in the AβOs groups versus the control groups（P<0.05）and in the WT/AβOs group versus the Ndfip1 Tg/AβOs group（P<0.05）.Conclusion:1.Ndfip1 overexpression antagonizes AβOs-induced neuronal toxicity,prevents neuronal damage,and promotes neuronal survival.2.Ndfip1 overexpression downregulates DMT1 expression,which may aid in maintaining intracellular iron homeostasis and suppressing ROS production and AβOs-induced neuronal apoptosis.3.Ndfip1 overexpression discreases caspase-3 activity,which may suppress AβOs-induced neuronal apoptosis perhaps by inhibiting ERK1/2 activity.