Inhibition of choline acetyltransferase as a mechanism for cholinergic dysfunction induced by amyloid-β peptide oligomers

Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.     

Deregulation of excitatory neurotransmission underlying synapse failure in Alzheimer's disease

Alzheimer′s disease (AD) is the most common form of dementia in the elderly. Memory loss in AD is increasingly attributed to soluble oligomers of the amyloid‐β peptide (AβOs), toxins that accumulate in AD brains and target particular synapses. Glutamate receptors appear to be centrally involved in synaptic targeting by AβOs. Once bound to neurons, AβOs dysregulate the activity and reduce the surface expression of both N‐methyl‐d ‐aspartate (NMDA) and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propanoic acid (AMPA) types of glutamate receptors, impairing signaling pathways involved in synaptic plasticity. In the extracellular milieu, AβOs promote accumulation of the excitatory amino acids, glutamate and d ‐serine. This leads to overactivation of glutamate receptors, triggering abnormal calcium signals with noxious impacts on neurons. Here, we review key findings linking AβOs to deregulated glutamate neurotransmission and implicating this as a primary mechanism of synapse failure in AD. We also discuss strategies to counteract the impact of AβOs on excitatory neurotransmission. In particular, we review evidence showing that inducing neuronal hyperpolarization via activation of inhibitory GABA_A receptors prevents AβO‐induced excitotoxicity, suggesting that this could comprise a possible therapeutic approach in AD.     

Calpain cleavage and inactivation of the sodium calcium exchanger‐3 occur downstream of Aβ in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β‐amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase‐3, activity is a prominent feature of AD brain. In addition, we observe increased calpain‐mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain‐dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.     

Ginkgolides protect against amyloid-β1–42-mediated synapse damage in vitro

Background

The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ_1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ_1–42 were examined.

Results

The addition of Aβ_1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ_1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ_1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ_1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ_1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ_1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E₂.

Conclusion

Pre-treatment with ginkgolides A or B protects neurons against Aβ_1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E₂, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.     

Resilin and chitinous cuticle form a composite structure for energy storage in jumping by froghopper insects

Background

Erythropoietin (EPO) improves cognition of human subjects in the clinical setting by as yet unknown mechanisms. We developed a mouse model of robust cognitive improvement by EPO to obtain the first clues of how EPO influences cognition, and how it may act on hippocampal neurons to modulate plasticity.

Results

We show here that a 3-week treatment of young mice with EPO enhances long-term potentiation (LTP), a cellular correlate of learning processes in the CA1 region of the hippocampus. This treatment concomitantly alters short-term synaptic plasticity and synaptic transmission, shifting the balance of excitatory and inhibitory activity. These effects are accompanied by an improvement of hippocampus dependent memory, persisting for 3 weeks after termination of EPO injections, and are independent of changes in hematocrit. Networks of EPO-treated primary hippocampal neurons develop lower overall spiking activity but enhanced bursting in discrete neuronal assemblies. At the level of developing single neurons, EPO treatment reduces the typical increase in excitatory synaptic transmission without changing the number of synaptic boutons, consistent with prolonged functional silencing of synapses.

Conclusion

We conclude that EPO improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks. These mechanisms of action of EPO have to be further exploited for treating neuropsychiatric diseases.     

Beta-amyloid, neuronal death and Alzheimer's disease

Alzheimer's disease (AD) is a common neurodegenerative disease that affects cognitive function in the elderly. Large extracellular beta-amyloid (Abeta) plaques and tau-containing intraneuronal neurofibrillary tangles characterize AD from a histopathologic perspective. However, the severity of dementia in AD is more closely related to the degree of the associated neuronal and synaptic loss. It is not known how neurons die and synapses are lost in AD; the current review summarizes what is known about this issue. Most evidence indicates that amyloid precursor protein (APP) processing is central to the AD process. The Abeta in plaques is a metabolite of the APP that forms when an alternative (beta-secretase and then gamma-secretase) enzymatic pathway is utilized for processing. Mutations of the APP gene lead to AD by influencing APP metabolism. One leading theory is that the Abeta in plaques leads to AD because Abeta is directly toxic to the adjacent neurons. Other theories advance the notion that neuronal death is triggered by intracellular events that occur during APP processing or by extraneuronal preplaque Abeta oligomers. Some investigators speculate that in many cases there is a more general disorder of protein processing in neurons that leads to cell death. In the later models, Abeta plaques are a byproduct of the disease process, rather than the direct cause of neuronal death. A direct correlation between Abeta plaque burden and neuronal (or synaptic) loss should occur in AD if Abeta plaques cause AD through a direct toxic effect. However, histopathologic studies indicate that the correlation between Abeta plaque burden and neuronal (or synaptic) loss is poor. We conclude that APP processing and Abeta formation is important to the AD process, but that neuronal alterations that underlie symptoms of AD are not due exclusively to a direct toxic effect of the Abeta deposits that occur in plaques. A more general problem with protein processing, damage due to the neuron from accumulation of intraneuronal Abeta or extracellular, preplaque Abeta may also be important as underlying factors in the dementia of AD.     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ(25-35) peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4±4.7% of the observed mitochondria were motile, with 21.0±1.3% moving anterograde and 15.4±3.4% moving retrograde and the average speed of movement was 12.1±1.8μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4±2.6% (P<0.032), as were those moving anterograde (10.1±2.6%, P<0.016) relative to PBS-treated neurons, suggesting that the Aβ(25-35) peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9±1.9μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.     

Alzheimer's disease amyloid β-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils

Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aβ proteins and their aggregation states or fibrillar structure.     

DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase‐β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild‐type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild‐type control mice, Polβ heterozygous (Polβ^+/?), and 3xTgAD mice, 3xTgAD/Polβ^+/? mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polβ^+/? and 3xTgAD mice, and by over 60% in the 3xTgAD/Polβ^+/? mice compared to wild‐type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ^+/? mice compared to 3xTgAD mice. Levels of amyloid β‐peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ^+/? mice, and cultured Polβ‐deficient neurons exhibited increased vulnerability to Aβ‐induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.     

ApoE isoform-dependent changes in hippocampal synaptic function

Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Aβ) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Aβ species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Aβ oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Aβ monomer or Aβ fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Aβ oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Aβ oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Aβ oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ_25-35 peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4 ± 4.7% of the observed mitochondria were motile, with 21.0 ± 1.3% moving anterograde and 15.4 ± 3.4% moving retrograde and the average speed of movement was 12.1 ± 1.8 μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4 ± 2.6% (P < 0.032), as were those moving anterograde (10.1 ± 2.6%, P < 0.016) relative to PBS-treated neurons, suggesting that the Aβ_25-35 peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9 ± 1.9 μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.     

Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia

Background

A mutation in the BRI2/ITM2b gene causes familial Danish dementia (FDD). BRI2 is an inhibitor of amyloid-?? precursor protein (APP) processing, which is genetically linked to Alzheimer??s disease (AD) pathogenesis. The FDD mutation leads to a loss of BRI2 protein and to increased APP processing. APP haplodeficiency and inhibition of APP cleavage by ??-secretase rescue synaptic/memory deficits of a genetically congruous mouse model of FDD (FDD_KI). ??-cleavage of APP yields the ??-carboxyl-terminal (??-CTF) and the amino-terminal-soluble APP?? (sAPP??) fragments. ??-secretase processing of ??-CTF generates A??, which is considered the main cause of AD. However, inhibiting A?? production did not rescue the deficits of FDD_KI mice, suggesting that sAPP??/??-CTF, and not A??, are the toxic species causing memory loss.

Results

Here, we have further analyzed the effect of ??-secretase inhibition. We show that treatment with a ??-secretase inhibitor (GSI) results in a worsening of the memory deficits of FDD_KI mice. This deleterious effect on memory correlates with increased levels of the ??/??-CTFs APP fragments in synaptic fractions isolated from hippocampi of FDD_KI mice, which is consistent with inhibition of ??-secretase activity.

Conclusion

This harmful effect of the GSI is in sharp contrast with a pathogenic role for A??, and suggests that the worsening of memory deficits may be due to accumulation of synaptic-toxic ??/??-CTFs caused by GSI treatment. However, ??-secretase cleaves more than 40 proteins; thus, the noxious effect of GSI on memory may be dependent on inhibition of cleavage of one or more of these other ??-secretase substrates. These two possibilities do not need to be mutually exclusive. Our results are consistent with the outcome of a clinical trial with the GSI Semagacestat, which caused a worsening of cognition, and advise against targeting ??-secretase in the therapy of AD. Overall, the data also indicate that FDD_KI is a valuable mouse model to study AD pathogenesis and predict the clinical outcome of therapeutic agents for AD.     

Transthyretin and the brain re-visited: Is neuronal synthesis of transthyretin protective in Alzheimer's disease?

Background

Previously we reported 1 μM synthetic human amyloid beta_1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.

Results

Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ_1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC₅₀) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ_1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.

Conclusions

Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.     

Andrographolide reduces cognitive impairment in young and mature AβPPswe/PS-1 mice

Alzheimer’s disease (AD) is a neurodegenerative disorder in which the amyloid-β (Aβ) oligomers are a key factor in synaptic impairment and in spatial memory decline associated with neuronal dysfunction. This impairment includes synaptic failure associated with the loss of synaptic proteins that contribute to AD progression. Interestingly, the use of natural compounds is an emergent conceptual strategy in the search for drugs with therapeutic potentials for treating neurodegenerative disorders. In the present study, we report that andrographolide (ANDRO), which is a labdane diterpene extracted from Andrographis paniculata, increases slope of field excitatory postsynaptic potentials (fEPSP) in the CA1 region of hippocampal slices and inhibits long-term depression (LTD), protecting the long-term potentiation (LTP) against the damage induced by Aβ oligomers in vitro, most likely by inhibiting glycogen synthase kinase-3β (GSK-3β). Additionally, ANDRO prevents changes in neuropathology in two different age groups (7- and 12-month-old mice) of an AβPPswe/PS-1 Alzheimer’s model. ANDRO reduces the Aβ levels, changing the ontogeny of amyloid plaques in hippocampi and cortices in 7-month-old mice, and reduces tau phosphorylation around the Aβ oligomeric species in both age groups. Additionally, we observed that ANDRO recovers spatial memory functions that correlate with protecting synaptic plasticity and synaptic proteins in two different age groups. Our results suggest that ANDRO could be used in a potential preventive therapy during AD progression.     

Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Mediates Neuronal Aβ42 Uptake and Lysosomal Trafficking

Background

Alzheimer''s disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-β 42 (Aβ42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Aβ42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Aβ42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Aβ42 through LRP1 is lacking.

Methodology/Principal Findings

Here we show that LRP1 endocytic function is required for neuronal Aβ42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Aβ42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Aβ42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Aβ42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Aβ42 is associated with increased cellular toxicity.

Conclusions/Significance

These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Aβ42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Aβ metabolism.     

Abnormal dendritic calcium activity and synaptic depotentiation occur early in a mouse model of Alzheimer’s disease

Background

Alzheimer’s disease (AD) is characterized by amyloid deposition, tangle formation as well as synapse loss. Synaptic abnormalities occur early in the pathogenesis of AD. Identifying early synaptic abnormalities and their underlying mechanisms is likely important for the prevention and treatment of AD.

Methods

We performed in vivo two-photon calcium imaging to examine the activities of somas, dendrites and dendritic spines of layer 2/3 pyramidal neurons in the primary motor cortex in the APPswe/PS1dE9 mouse model of AD and age-matched wild type control mice. We also performed calcium imaging to determine the effect of Aβ oligomers on dendritic calcium activity. In addition, structural and functional two-photon imaging were used to examine the link between abnormal dendritic calcium activity and changes in dendritic spine size in the AD mouse model.

Results

We found that somatic calcium activities of layer 2/3 neurons were significantly lower in the primary motor cortex of 3-month-old APPswe/PS1dE9 mice than in wild type mice during quiet resting, but not during running on a treadmill. Notably, a significantly larger fraction of apical dendrites of layer 2/3 pyramidal neurons showed calcium transients with abnormally long duration and high peak amplitudes during treadmill running in AD mice. Administration of Aβ oligomers into the brain of wild type mice also induced abnormal dendritic calcium transients during running. Furthermore, we found that the activity and size of dendritic spines were significantly reduced on dendritic branches with abnormally prolonged dendritic calcium transients in AD mice.

Conclusion

Our findings show that abnormal dendritic calcium transients and synaptic depotentiation occur before amyloid plaque formation in the motor cortex of the APPswe/PS1dE9 mouse model of AD. Dendritic calcium transients with abnormally long durations and high amplitudes could be induced by soluble Aβ oligomers and contribute to synaptic deficits in the early pathogenesis of AD.

     

Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer's disease: implications to mitochondria-targeted antioxidant therapeutics

Synaptic pathology and mitochondrial oxidative damage are early events in Alzheimer's disease (AD) progression. Loss of synapses and synaptic damage are the best correlates of cognitive deficits found in AD patients. Recent research on amyloid beta (Aβ) and mitochondria in AD revealed that Aβ accumulates in synapses and synaptic mitochondria, leading to abnormal mitochondrial dynamics and synaptic degeneration in AD neurons. Further, recent studies using live-cell imaging and primary neurons from amyloid beta precursor protein (AβPP) transgenic mice revealed reduced mitochondrial mass, defective axonal transport of mitochondria and synaptic degeneration, indicating that Aβ is responsible for mitochondrial and synaptic deficiencies. Tremendous progress has been made in studying antioxidant approaches in mouse models of AD and clinical trials of AD patients. This article highlights the recent developments made in Aβ-induced abnormal mitochondrial dynamics, defective mitochondrial biogenesis, impaired axonal transport and synaptic deficiencies in AD. This article also focuses on mitochondrial approaches in treating AD, and also discusses latest research on mitochondria-targeted antioxidants in AD. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers

Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. AβOs have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of AβOs. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by AβOs in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser(845), which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of AβOs, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by AβOs in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD.     

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Background

Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na⁺ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.

Results

Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na⁺ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Na_v1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Na_v1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.

Conclusion

Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Na_v1.3 subunit, and the function of the Na_v1.3 channel is regulated by PKC.     

The protection of novel 2-arylethenylquinoline derivatives against impairment of associative learning memory induced by neural Aβ in <Emphasis Type="Italic">C. elegans</Emphasis> Alzheimer’s disease model

Cerebral deposition of amyloid β-peptide (Aβ), a fundamental feature of Alzheimer’s disease (AD), damages the neurocytes and impairs the cognition functions and associative learning memory of AD patients. A series of novel 2-arylethenylquinoline derivatives were synthesized and evaluated in our previous study, which inhibited Aβ aggregation in vitro effectively at the concentration of 20 μmol/L and exhibited high antioxidant activity. In order to verify the capacity of anti-AD in vivo, the transgenic Caenorhabditis elegans (C. elegans) strain CL2355 expressing neural Aβ was employed as the AD model to investigate the neuroprotective activity of seven high-potential compounds (4a1, 4a2, 4b1, 4b2, 4c1, 4c2, 4c3) selected from those derivatives. Learning memory associated chemotaxis assay was performed to evaluate the neural repairment capacity. The underlying mechanism was investigated by mRNA analysis of Aβ gene and heat shock protein genes (hsp-16.1 and hsp-16.2) and Western blot of Aβ. Our data indicated that among seven tested compound, 4b1 and 4c2 reduced Aβ-induced stress, suppressed the expression of neural Aβ monomers and toxic oligomers, and recovered the damaged associative learning memory in C. elegans AD model. These findings further confirmed their potentials to become valuable agents for AD therapy.