A distinct subfraction of Aβ is responsible for the high‐affinity Pittsburgh compound B‐binding site in Alzheimer's disease brain

The positron emission tomography (PET) ligand ¹¹C‐labeled Pittsburgh compound B (PIB) is used to image β‐amyloid (Aβ) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human‐sequence Aβ in amyloid precursor protein transgenic mice and non‐human primates bind very little PIB. The high stoichiometry of PIB:Aβ binding in human AD suggests that the PIB‐binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, ³H‐PIB was employed to track purification of the PIB‐binding site in > 90% yield from frontal cortical tissue of autopsy‐diagnosed AD subjects. The purified PIB‐binding site comprises a distinct, highly insoluble subfraction of the Aβ in AD brain with low buoyant density because of the sodium dodecyl sulfate‐resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aβ complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human‐specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues.

     

Expression of the synaptic exocytosis‐regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.

     

Overactivation of NR2B‐containing NMDA receptors through entorhinal–hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion

Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase‐3β at Ser 9 in the ipsilateral hippocampus. These MCAO‐induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N‐methyl‐d ‐aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B‐containing NMDARs through entorhinal–hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase‐3β is an important protein kinase involved in NMDARs‐mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B‐containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post‐stroke dementia.

     

Impaired clustered protocadherin‐α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice

Clustered protocadherins (cPcdhs) comprising cPcdh‐α, ‐β, and ‐γ, encode a large family of cadherin‐like cell‐adhesion molecules specific to neurons. Impairment of cPcdh‐α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh‐α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh‐α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh‐α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh‐α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

     

P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway

Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium‐evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4‐ and 7‐fold increase in potassium‐evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal‐dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability.

     

Tau‐targeting passive immunization modulates aspects of pathology in tau transgenic mice

Immunization is increasingly recognized as a suitable therapeutic avenue for the treatment of neurological diseases such as Alzheimer's disease and other tauopathies. Tau is a key molecular player in these conditions and therefore represents an attractive target for passive immunization approaches. We performed such an approach in two independent tau transgenic mouse models of tauopathy, K369I tau transgenic K3 and P301L tau transgenic pR5 mice. The antibodies we used were either specific for full‐length tau or tau phosphorylated at serine 404 (pS404), a residue that forms part of the paired helical filament (PHF)‐1 phosphoepitope that characterizes tau neurofibrillary tangles in tauopathies. Although both pS404 antibodies had a similar affinity, they differed in isotype, and only passive immunization with the IgG2a/κ pS404‐specific antibody resulted in a lower tangle burden and reduced phosphorylation of tau at the PHF1 epitope in K3 mice. In pR5 mice, the same antibody led to a reduced phosphorylation of the pS422 and PHF1 epitopes of tau. In addition, histological sections of the hippocampal dentate gyrus of the immunized pR5 mice displayed reduced pS422 staining intensities. These results show that passive immunization targeting tau can modulate aspects of tau pathology in tau transgenic mouse models, in an antibody isotype‐specific manner.

     

Knockdown of phosphotyrosyl phosphatase activator induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down‐regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase‐3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl‐xl and Bcl‐2 protein levels. Over‐expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2A_C) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen‐activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over‐expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA‐induced cell death.

     

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP₇₇₀, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP₆₉₅ (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.

     

Amyloid precursor proteins are constituents of the presynaptic active zone

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface‐localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock‐out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.

     

Dysregulation of leptin signaling in Alzheimer disease: evidence for neuronal leptin resistance

Leptin signaling has received considerable attention in the Alzheimer disease (AD) field. Within the past decade, the peptide hormone has been demonstrated to attenuate tau hyperphosphorylation in neuronal cells and to be modulated by amyloid‐β. Moreover, a role in neuroprotection and neurogenesis within the hippocampus has been shown in animal models. To further characterize the association between leptin signaling and vulnerable regions in AD, we assessed the profile of leptin and the leptin receptor in AD and control patients. We analyzed leptin levels in CSF, and the concentration and localization of leptin and leptin receptor in the hippocampus. Significant elevations in leptin levels in both CSF and hippocampal tissue of AD patients, compared with age‐matched control cases, indicate a physiological up‐regulation of leptin in AD. However, the level of leptin receptor mRNA decreased in AD brain and the leptin receptor protein was localized to neurofibrillary tangles, suggesting a severe discontinuity in the leptin signaling pathway. Collectively, our results suggest that leptin resistance in the hippocampus may play a role in the characteristic changes associated with the disease. These findings are the first to demonstrate such dysregulated leptin‐signaling circuitry and provide novel insights into the possible role of aberrant leptin signaling in AD.

     

Sulforaphane enhances temozolomide‐induced apoptosis because of down‐regulation of miR‐21 via Wnt/β‐catenin signaling in glioblastoma

Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.

     

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.

     

Is the mTOR‐signalling cascade disrupted in Schizophrenia?

The mammalian target of rapamycin (mTOR) signalling cascade is involved in the intracellular regulation of protein synthesis, specifically for proteins involved in controlling neuronal morphology and facilitating synaptic plasticity. Research has revealed that the activity of the mTOR cascade is influenced by several extracellular and environmental factors that have been implicated in schizophrenia. Therefore, there is reason to believe that one of the downstream consequences of dysfunction or hypofunction of these factors in schizophrenia is disrupted mTOR signalling and hence impaired protein synthesis. This results in abnormal neurodevelopment and deficient synaptic plasticity, outcomes which could underlie some of the positive, negative and cognitive symptoms of schizophrenia. This review will discuss the functional roles of the mTOR cascade and present evidence in support of a novel mTOR‐based hypothesis of the neuropathology of schizophrenia.

     

Nicotine induces dendritic spine remodeling in cultured hippocampal neurons

Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR‐induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre‐synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro‐β‐erythroidine, an antagonist of α4β2* nAChRs, but not by α‐bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2‐amino‐5‐phosphonovaleric acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, antagonists of NMDA‐ and AMPA‐type glutamate receptors, blocked the nicotine‐induced spine remodeling. In addition, nicotine exerted full spine‐enlarging response in the post‐synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre‐treatment with nicotine enhanced the Ca²⁺‐response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre‐synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function.

     

Parkin reverses TDP‐43‐induced cell death and failure of amino acid homeostasis

The E3 ubiquitin ligase Parkin plays a central role in the pathogenesis of many neurodegenerative diseases. Parkin promotes specific ubiquitination and affects the localization of transactivation response DNA‐binding protein 43 (TDP‐43), which controls the translation of thousands of mRNAs. Here we tested the effects of lentiviral Parkin and TDP‐43 expression on amino acid metabolism in the rat motor cortex using high frequency ¹³C NMR spectroscopy. TDP‐43 expression increased glutamate levels, decreased the levels of other amino acids, including glutamine, aspartate, leucine and isoleucine, and impaired mitochondrial tricarboxylic acid cycle. TDP‐43 induced lactate accumulation and altered the balance between excitatory (glutamate) and inhibitory (GABA) neurotransmitters. Parkin restored amino acid levels, neurotransmitter balance and tricarboxylic acid cycle metabolism, rescuing neurons from TDP‐43‐induced apoptotic death. Furthermore, TDP‐43 expression led to an increase in 4E‐BP levels, perhaps altering translational control and deregulating amino acid synthesis; while Parkin reversed the effects of TDP‐43 on the 4E‐BP signaling pathway. Taken together, these data suggest that Parkin may affect TDP‐43 localization and mitigate its effects on 4E‐BP signaling and loss of amino acid homeostasis.

     

Optineurin suppression causes neuronal cell death via NF‐κB pathway

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss‐of‐function mutation. Wild‐type optineurin (OPTN^WT) suppresses nuclear factor‐kappa B (NF‐κB) activity, but the ALS‐causing mutant OPTN is unable to suppress NF‐κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF‐κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF‐κB activity was increased in OPTN‐knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTN^WT or OPTN^E50K with intact NF‐κB‐suppressive activity, but not overexpression of ALS‐related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF‐κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF‐κB activation is the pathogenic mechanism underlying OPTN mutation‐related ALS.