Amyloid Beta-mediated KIF5A Deficiency Disrupts Anterograde Axonal Mitochondrial Movement

Background:Alzheimer’s disease(AD)is a common neurodegenerative disease characterized by cognitive decline,in particular progressive memory loss.Alzheimer’ s disease has been identified as a protein misfolding disease,caused by plaque accumulation of abnormally folded amyloid beta protein and tau protein in the brain,which cause synaptic dysfunction and neuron losses.The cause of Alzheimer’ s disease is poorly understood till now.The amyloid hypothesis,which is widely accepted,po_s tulated that extracellular amyloid beta,especially Aβ42,deposits are the fundamental cause of the disease.The hypothesis further suggests that the amyloid precursor protein(APP)gene,the presenilin 1(PS1)gene and the presenilin 2(PS2)gone mutation are important causes of Aβ 42 production.However,the study found that there is no APP or PS gene mutation in patients with sporadic AD,so the hypothesis still has certain limitations.In recent years,a large number of studies suggest that mitochondrial dysfunction is one of the important pathological changes of AD.Mitochondrial dysfunction can occur in AD patients early,and mitochondrial damage with AD progression is deteriorating and ultimately affects synaptic and neuronal function.Based on this,Swerdlow et al.proposed the mitochondrial cascade hypothesis for sporadic AD.The hypothesis is that mitochondrial dysfunction is the initial event leading to AD.Aβ deposition,tau phosphorylation,and neuronal degeneration are all based on mitochondrial dysfunction,which further aggravates mitochondrial dysfunction.Although the mitochondrial cascade hypothesis also has its inherent limitations,it provides a new idea for the study of AD.线Mitochondria are important organelles in neurons.In addition to providing the ATP required to maintain neuronal function,mitochondria maintain synaptic plasticity by providing energy,regulating synaptic calcium homeostasis,mediating dialogue between synapses/neuronal nuclei,and regulating oxygen free radical levels.And synaptic transmission plays a key role in achieving neuronal function.It is worth noting that the mitochondrial synthesis site is the neuronal cell body,after which the neuron delivers the mitochondria to the synaptic site.Therefore,normal mitochondrial transport is essential for maintaining synaptic function;mitochondrial transport disorders are an important cause of neuronal damage,especially synaptic function.In recent years,the relationship between neuronal mitochondrial transport disorders and neuronal damage in AD has been receiving attention.A large number of studies have shown that mitochondrial transport disorders,especially mitochondria,are one of the causes of synaptic dysfunction.However,the specific molecular mechanisms by which neurons are transported in the axial direction are not yet known.Kinesin superfamily proteins(KIFs)and cytoplasmic dynein are the major types of microtubule-dependent motor proteins,which are essential molecular machineries for intracellular transport.KIF5A is a key isoform of kinesin-1,which is a key molecular machinery in facilitating anterograde axonal mitochondrial transport.Previous studies have shown that down-regulation of KIF5A expression may be responsible for the disruption of anterograde axonal mitochondrial transport in AD.The purpose of this study is to explore the specific molecular mechanism of KIF5A leading to neuronal mitochondrial axial transport disorders in AD state by pathology and molecular biology techniques.Aims:1.To define changes of proteins revolved in mitochondrial axonal transport in AD;2.To find the possible mechanism of KIF5A decline in AD state;3.To clarify the effect of KIF5A downregulation on the anterograde axonal mitochondrial transport;4.To explore the effect of KIF5A restoration on axonal mitochondrial transport and neuronal function in AD state.Methods:1.Mice:8 and 12 months old nonTg and 5xFAD mice of mixed genders were used in the experiments.Day 0-1 pups from C57BL/6J strain mice were used for primary neuron culture.2 Human Samples:Frozen brain samples and paraffin-embedded brain slices were requested from UT Southwestern Medical Center ADC Neuropathology Core,supported by ADC grant(AG12300)under a protocol approved by The UT Southwestern Medical Center with informed consent from all subjects and the study adhered to the Declaration of Helsinki principles.3 Primary neurons culture:Cortices or hippocampi were dissected from Day 0 pups from C57BL/6J strain mice.Tissues were digested in trypsin and primary neurons were cultured in neuron culture medium(Neurobasal A with B27 supplement,L-glutamine,penicillin,and streptomycin).Mitochondria motility measurement was conducted on neurons with density of 0.1 × 105/cm2.The samples for Western Blot were collected from neurons with density of 1×105/cm2.4.Immunoblotting:Western Blot was used to detect changes in neuronal mitochondrial-related protein expression levels.5.Immunostaining:Brain tissues from 8 and 12 months old nonTg and 5× FAD mice were used to perform immunostaining,which to6.Generation of Lentivirus expressing mitochondria targeted DsRed:HEK293T cells(ATCC)were transiently cotransfected with lentivirus vector carrying mitochondria targeted DsRed by using calcium phosphate precipitation method.The lentivirus were collected from the culture medium and concentrated with Lenti-X Concentrator(Clontech).7.KIF5A knocking down in mouse primary neurons:Lentivirus-expressing shRNA targeted to mouse KIF5A was packaged with lentivirus shRNA construct.Mouse primary neurons were cultured for 3 days before infection with lentivirus.Neurons were treated and collected for experiments after a further 7 days in culture.8.KIF5A Overexpression in mouse primary neurons:Human KIF5A was inserted into lentivirus vector with human polyubiquitin promoter-C.Lentivirus containing KIF5A were packaged and applied on primary neurons similar as shRNA lentiviral vector.Mouse primary neurons were cultured for 3 days before infection with lentivirus at a m.o.i.of 5.Neurons were collected for experiments after 7 days infection.9.Oligomeric Aβ preparation:The peptide solution was resuspended in cold HAM’S F-12 to 100 u M and immediately vortexed for 30 s.The solution was then incubated at 4 0 C for 24 h.Oligomeric A β(1μM)was applied on neurons for 24 h before cell collection.10.Mitochondria motility measurement:Mouse primary neurons were infected by lentivirus expressing mtDsRed to visualized mitochondria.Time-lapse images were captured every 5 seconds for a total of 3 minutes under 40X oil objective on a Nikon inverted fluorescent microscope with an on-stage incubator(37℃,5%C02).The velocity of all mobile mitochondria was measured by using tracking system integrated in Nikon NIS Advanced Research software.11.Statistical analysisStatistical comparisons were performed using GraphPad Prism 5 software.One-way ANOVA followed by Bonferroni post hoc analysis,or unpaired two-way Student’ s t test were applied in data analysis.Numbers of replicates and P value are stated in each figure legend.All data were expressed as the mean±s.e.m.Significance was concluded when the P value was less than 0.05.Results:1.KIF5A is down-regulated in temporal lobe tissue of AD patients.Western blotting was used to detect the expression levels of mitochondrial transport-related proteins KIF5A,KIF5B,KIF5C,Dynein,Syntabulin,Mirol and mitochondrial marker Tom40 in the temporal and cerebellar tissues of patients with AD and non-AD subjects.KIF5A was found decreased significantly in temporal lobe,which is the AD-sensitive brain aera and KIF5B showed a slight decrease in the temporal lobe tissue,but no change was observed in the cerebellar tissue.Further correlation analysis found that the decrease in KIF5A expression levels was independent of age and time of death.2.The decrease in KIF5A is closely related to Aβ toxicity.We used 5xFAD mice at 8 months and 12 months of age.Using immunoblotting and immunofluorescence,we found that KIF5A decreased significantly in mouse cortex,KIF5B also decreased slightly,while KIF5C,Dynein,Syntabulin and other proteins did not change.Mirol and Tom40 only decreased in the cortex of 12-month-old mice.Further detection of Aβ-treated primary neurons by immunoblotting revealed that KIF5A expression levels were still significantly decreased,while KIF5B,KIF5C,Dynein,Syntabulin and other protein expression levels were not significantly changed.Mirol expression levels were slightly downregulated.3.Downregulation of KIF5A expression levels impeded the smooth transport of neuronal mitochondria.We found that down-regulation of KIF5A expression reduced the number and ratio of neuronal mitochondrial transport in the axial direction,while the rate of mitochondrial transport in the axial direction was significantly lower than that of the control group,while neuronal mitochondrial retrograde axonal mitochondrial transport was not affected.This change is similar to Aβ-mediated neuronal mitochondrial transport disorders.4.KIF5A overexpression effectively rescues neuronal mitochondrial axial transport disorders under Aβ conditions.A P treatment of KIF5A overexpression and normal control neurons,the results show that KIF5A overexpressing virus-treated neurons can effectively reduce the down-regulation of KIF5A expression induced by A p,and increase the proportion and rate of neuronal mitochondria transport in the axial direction.5.KIF5A overexpression effectively rescues neuronal damage caused by A p.Reconstruction of KIF5A expression level can effectively reduce the increase of oxygen free radicals in neurons induced by Aβ,and promote the formation of synapses.Conclusions:1.The expression level of KIF5A in temporal lobe tissue of AD patients decreased;2.The decrease in the expression level of KIF5A is closely related to the toxicity of Aβ;3.Down-regulation of KIF5A expression is one of the causes of neuronal mitochondrial axial transport disorder under AD conditions;4.Reconstruction of KIF5A expression level can effectively rescue the mitochondrial dysplasia of neurons under AD conditions and improve the function of neurons and synapses.