The 'toxic Aβ oligomer' hypothesis has attracted considerable attention among Alzheimer's disease researchers as a way of resolving the lack of correlation between deposited amyloid-β (Aβ) in amyloid plaques-in terms of both amount and location-and cognitive impairment or neurodegeneration. However, the lack of a common, agreed-upon experimental description of the toxic Aβ oligomer makes interpretation and direct comparison of data between different research groups impossible. Here we critically review the evidence supporting toxic Aβ oligomers as drivers of neurodegeneration and make some suggestions that might facilitate progress in this complex field.

The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes

https://www.cell.com/cell/pdf/S0092-8674(09)00446-2.pdf

Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

The human mitochondrial genome is extremely small compared with the nuclear genome, and mitochondrial genetics presents unique clinical and experimental challenges. Despite the diminutive size of the mitochondrial genome, mitochondrial DNA (mtDNA) mutations are an important cause of inherited disease. Recent years have witnessed considerable progress in understanding basic mitochondrial genetics and the relationship between inherited mutations and disease phenotypes, and in identifying acquired mtDNA mutations in both ageing and cancer. However, many challenges remain, including the prevention and treatment of these diseases. This review explores the advances that have been made and the areas in which future progress is likely.

/pdf/mitochondrial-dna-mutations-in-human-disease-361jhw3bud.pdf

Mitochondrial dna mutations in human disease

The mitochondrial respiratory chain has the crucial function of supplying the cell with energy in the form of ATP. Mutations affecting this chain can arise in mitochondrial or nuclear DNA and cause diseases known as mitochondrial encephalomyopathies. Because the rules of inheritance of mitochondrial and nuclear DNA differ considerably, these brain–muscle syndromes often have unpredictable clinical and genetic features.

https://www.nejm.org/doi/pdf/10.1056/NEJMra022567

Mitochondrial respiratory-chain diseases

s | 77 1Chonnam National University, Republic of Korea, 2Orygen Youth Health, Australia, 3Deakin University, Australia, 4Medical University of Vienna, Austria, 5University Hospital Jena, Germany Abstract Object: This study investigated the relationship between erythrocyte membrane fatty acid (FA) levels and the severity of symptoms of individuals at ultra-high risk(UHR) for psychosis. Methods: The study sample consisted of 80 neuroleptic-naïve UHR patients. Associations between baseline erythrocyte membrane FA levels, measured by gas chromatography, and scores on the Positive and Negative Syndrome Scale (PANSS), the Global Assessment of Functioning Scale, and the Montgomery–Asberg Depression Rating Scale (MADRS) were investigated. After correlation analysis in all participants, subjects were divided into three groups according to the predominance of positive or negative symptoms based on PANSS subscale scores; membrane FA levels in the three groups were then compared. Results: PANSS negative symptom scores were negatively correlated with two saturated FAs (myristic and margaric acids), one ω-9 monounsaturated FA (MUFA;nervonic acid), and oneω-3 polyunsaturated FA(PUFA; docosapentaenoic acid). Negative symptom scores were positively correlated with twoω-9 MUFAs(eicosenoic and erucic acids) and two ω-6 PUFAs (γ-linoleic and docosadienoic acids). PANSS positive symptom scores were correlated only with nervonic acid. No associations were observed between FAs and MADRS scores. In subjects with dominant negative symptoms, the sum of the ω-9 MUFAs and the ω-6:ω-3 FA ratio were both significantly higher than in those with dominant positive symptoms, whereas the sum of ω-3 PUFAs was significantly lower. Conclusions: Abnormalities in FA metabolism may contribute to the neurobiology of psychopathology in UHR individuals. In particular, membrane FA alterations may play a role in negative symptoms, which are primary psychopathological manifestations of schizophrenia-related disability.Object: This study investigated the relationship between erythrocyte membrane fatty acid (FA) levels and the severity of symptoms of individuals at ultra-high risk(UHR) for psychosis. Methods: The study sample consisted of 80 neuroleptic-naïve UHR patients. Associations between baseline erythrocyte membrane FA levels, measured by gas chromatography, and scores on the Positive and Negative Syndrome Scale (PANSS), the Global Assessment of Functioning Scale, and the Montgomery–Asberg Depression Rating Scale (MADRS) were investigated. After correlation analysis in all participants, subjects were divided into three groups according to the predominance of positive or negative symptoms based on PANSS subscale scores; membrane FA levels in the three groups were then compared. Results: PANSS negative symptom scores were negatively correlated with two saturated FAs (myristic and margaric acids), one ω-9 monounsaturated FA (MUFA;nervonic acid), and oneω-3 polyunsaturated FA(PUFA; docosapentaenoic acid). Negative symptom scores were positively correlated with twoω-9 MUFAs(eicosenoic and erucic acids) and two ω-6 PUFAs (γ-linoleic and docosadienoic acids). PANSS positive symptom scores were correlated only with nervonic acid. No associations were observed between FAs and MADRS scores. In subjects with dominant negative symptoms, the sum of the ω-9 MUFAs and the ω-6:ω-3 FA ratio were both significantly higher than in those with dominant positive symptoms, whereas the sum of ω-3 PUFAs was significantly lower. Conclusions: Abnormalities in FA metabolism may contribute to the neurobiology of psychopathology in UHR individuals. In particular, membrane FA alterations may play a role in negative symptoms, which are primary psychopathological manifestations of schizophrenia-related disability. PM487 Presynaptic protein Piccolo knockdown in the prefrontal cortex induces cognitive and emotional impairment in mice. Yoshiaki Miyamoto1, Ryo Inagaki1, Keiji Sato1, Shin-ichi Muramatsu2, Toshitaka Nabeshima3, Kyosuke Uno1, Atsumi Nitta1 1Department of Pharmaceutical Therapy and Neuropharmacology, Faculty of Pharmaceutical Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, JAPAN. 2Division of Neurology, Department of Medicine, Jichi Medial University, Shimotsuke 329–0498, JAPAN. 3Nabeshima Laboratory, Meijo University, Nagoya 468–8503, JAPAN. Abstract Piccolo, a protein of the cytomatrix at the active zone, is involved in the exocytosis and endocytosis of the synaptic vesicles at the presynaptic bouton. It is reported recently that the expression level of Piccolo-encoded PCLO mRNA and Piccolo itself changed in the brain of the bipolar disorder and schizophrenia patient. In the present study, therefore, to clarify the neurological function of Piccolo in the development of mental disorders, we generated Piccolo deficient animal by bilaterally microinjecting the adeno-associated virus vector containing PCLO miRNA into the prefrontal cortex of the mouse. In the electrophysiological analysis, the Piccolo knockdown mice were attenuated the paired-pulse facilitation and long-term potentiation in the prefrontal cortex. In the same prefrontal cortex, the phosphorylation level of synaptic vesicle binding protein Synapsin significantly decreased in the Piccolo knockdown mice, but the remarkable abnormalities were not observed on the shape of dendrite and spine and on the expression level of Synaptophysin, NMDAR1 and PSD-95 in the mice. In the behavioral analysis, the Piccolo knockdown mice showed decreased cognitive dysfunction including object recognition, spatial learning and working memory. Furthermore, the locomotor activity in the novel environment increased in the Piccolo knockdown mice, and its hyperlocomotion was improved by the treatment with atypical antipsychotic drug risperidone. Similarly, the impaired prepulse inhibition of the acoustic startle responses in the Piccolo knockdown mice was ameliorated by risperidone. These results suggest that Piccolo in the prefrontal cortex regulates the release of the neurotransmitter from the presynaptic membrane, and it consequently plays a functional role in the synaptic plasticity, cognitive memory and emotional activity. Furthermore, our observations indicate that the mouse with Piccolo deficient in the prefrontal cortex is useful animal model for the mental disorders such as the manic symptom of bipolar disorder or the positive symptom of schizophrenia.Piccolo, a protein of the cytomatrix at the active zone, is involved in the exocytosis and endocytosis of the synaptic vesicles at the presynaptic bouton. It is reported recently that the expression level of Piccolo-encoded PCLO mRNA and Piccolo itself changed in the brain of the bipolar disorder and schizophrenia patient. In the present study, therefore, to clarify the neurological function of Piccolo in the development of mental disorders, we generated Piccolo deficient animal by bilaterally microinjecting the adeno-associated virus vector containing PCLO miRNA into the prefrontal cortex of the mouse. In the electrophysiological analysis, the Piccolo knockdown mice were attenuated the paired-pulse facilitation and long-term potentiation in the prefrontal cortex. In the same prefrontal cortex, the phosphorylation level of synaptic vesicle binding protein Synapsin significantly decreased in the Piccolo knockdown mice, but the remarkable abnormalities were not observed on the shape of dendrite and spine and on the expression level of Synaptophysin, NMDAR1 and PSD-95 in the mice. In the behavioral analysis, the Piccolo knockdown mice showed decreased cognitive dysfunction including object recognition, spatial learning and working memory. Furthermore, the locomotor activity in the novel environment increased in the Piccolo knockdown mice, and its hyperlocomotion was improved by the treatment with atypical antipsychotic drug risperidone. Similarly, the impaired prepulse inhibition of the acoustic startle responses in the Piccolo knockdown mice was ameliorated by risperidone. These results suggest that Piccolo in the prefrontal cortex regulates the release of the neurotransmitter from the presynaptic membrane, and it consequently plays a functional role in the synaptic plasticity, cognitive memory and emotional activity. Furthermore, our observations indicate that the mouse with Piccolo deficient in the prefrontal cortex is useful animal model for the mental disorders such as the manic symptom of bipolar disorder or the positive symptom of schizophrenia. PM488 Activation of Galphaq proteins coupled with dopamine D1-like receptor and alpha1-adrenoceptor in rat brain membranes Yuji Odagaki, Masakazu Kinoshita, Toshio Ota Saitama Medical University, Japan Abstract Objectives: Previously, we reported 5-HT2A receptorand M1 muscarinic acetylcholine receptor (mAChR)-mediated Gαq activation in rat brain membranes (Eur J Pharmacol 726: 109–115, 2014). In this paper, it was noticed that Gαq proteins were also activated by dopamine and (-)-epinephrine, although these effects were much lower than that elicited by 5-HT or carbachol. In the present study, activation of Gαq coupled to dopaminergic and adrenergic receptor was pharmacologically characterized in rat brain membranes. Methods: Receptor-mediated activation of Gαq in rat brain membranes was assessed by guanosine-5’-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation assay (Eur J Pharmacol 726: 109–115, 2014), with minor modifications. Results: In cerebral cortical membranes, dopamine and (-)-epinephrine stimulated specific [35S]GTPγS binding to Gαq in a concentration-dependent manner, with EC50 values of 65 and 0.46 μM, to the maximal percent increase over basal binding of 99 and 70 %, respectively. The responses in hippocampus and striatum were lower than those in cerebral cortex for either compound. Pharmacological characterization using a series of dopaminergic and 

/pdf/pm487-presynaptic-protein-piccolo-knockdown-in-the-3xx1lefqje.pdf

PM487. Presynaptic protein Piccolo knockdown in the prefrontal cortex induces cognitive and emotional impairment in mice.

The current clinical trials of gene therapy for Parkinson's disease (PD) are based on three strategies. 1. To restore the local production of dopamine by introducing genes associated with dopamine-synthesizing enzymes into the putamen. 2. To protect nigrostriatal projection by delivering the neurturin gene, a trophic factor for dopaminergic neurons, both in the putamen and the substantia nigra. 3. To modulate the neural activity by transducing the subthalamic nucleus with vectors expressing glutamic acid decarboxylase. A phase I clinical study was initiated in 2007 to determine the safety of intra-putaminal infusion of a recombinant adeno-associated virus (AAV) vector encoding aromatic (L)-amino acid decarboxylase (AADC). All six patients enrolled in the trial showed improvements from baseline in the Unified Parkinson's Disease Rating Scale motor scores in the OFF medication state at 36 months after treatment. Although this trial was a small, open-label study and the use of a non-blinded, uncontrolled analysis limits interpretation, the efficacy outcomes are encouraging and indicate that the AAV vector-mediated gene transfer of AADC may benefit advanced PD patients. A similar approach, delivering AAV vector carrying AADC gene into the putamen ameliorated the symptoms in children with AADC deficiency.

[Gene therapy for Parkinson's disease].

Neural transplantation, as a treatment for advanced Parkinson's disease (PD), has been studied for more than a decade due to the potential replacement of degenerated dopaminergic (DA) neurons. Several open-label studies on implantation of fetal nigral neurons revealed improvement in motor functions. However, the benefits were incomplete in double-blind trials. Progressive neural or embryonic stem (ES) cell research has raised hopes of creating novel cell replacement therapies for PD. DA neurons have been efficiently produced from primate ES cells in astrocyte-conditioned medium. Transplantation of neuronal stem cells derived from primate ES cells into a primate model of PD restored striatal DA function, suggesting ES cells are suitable donor cells.

[ES cell therapy for Parkinson's disease].

Amyloid-β (Aβ) and tau pathologies are important factors leading to neurodegeneration in Alzheimer's disease (AD); however the molecular mechanisms that link these pathologies remain unclear. Assuming that important though as yet unidentified factors inhibit/accelerate tau pathology and neuronal cell death under amyloid pathology, we sought to isolate and identify tau-interacting proteins from mouse brains with or without amyloid pathology. Among the proteins that were identified, we focused on protein arginine methyltransferase 8 (PRMT8), which interacts with tau specifically in the absence of amyloid pathology. To investigate the role of PRMT8 in the pathogenesis of AD, we conducted Prmt8 gene deletion and overexpression experiments in AppNL-G-F/MAPT double-knock-in (dKI) mice and analyzed the resulting pathological alterations. PRMT8-knockout did not alter the AD pathology in dKI mice, whereas PRMT8-overexpression promoted tau phosphorylation, neuroinflammation, and vacuole degeneration. To evaluate if such a PRMT8-induced vacuole degeneration depends on tau pathology, PRMT8 was overexpressed in tau-KO mice, which were consequently found to exhibit vacuole degeneration. In addition, proteomic analyses showed that PRMT8 overexpression facilitated the arginine methylation of vimentin. Abnormal protein methylation could be involved in PRMT8-induced brain pathologies. Taken together, PRMT8 may play an important role in the formation of tau pathology and vacuole degeneration.

Tau-binding protein PRMT8 facilitates vacuole degeneration in the brain.

Gene therapy using adeno-associated virus (AAV) is a rapidly developing technology with widespread treatment potential. AAV2 vectors injected directly into the brain by stereotaxic brain surgery have shown good results in treating aromatic l-amino acid decarboxylase deficiency. Moreover, gene therapy using the AAV9 vector, which crosses the blood-brain barrier, has been performed in more than 2000 patients worldwide as a disease-modifying therapy for spinal muscular atrophy. AAV vectors have been applied to the development of gene therapies for various pediatric diseases. Gene therapy trials for hemophilia and ornithine transcarbamylase deficiency are underway. Clinical trials are planned for glucose transporter I deficiency, Niemann-Pick disease type C, and spinocerebellar ataxia type 1. The genome of AAV vectors is located in the episome and is rarely integrated into chromosomes, making the vectors safe. However, serious adverse events such as hepatic failure and thrombotic microangiopathy have been reported, and ongoing studies are focusing on developing more efficient vectors to reduce required dosages. 

Shin-ichi Muramatsu

Papers

PM487. Presynaptic protein Piccolo knockdown in the prefrontal cortex induces cognitive and emotional impairment in mice.

[Gene therapy for Parkinson's disease].

[ES cell therapy for Parkinson's disease].

Tau-binding protein PRMT8 facilitates vacuole degeneration in the brain.

Adeno-associated virus vector-based gene therapies for pediatric diseases.