Hasanuz Zaman , Anthony Holland. Kinetics and molecular docking studies of fucosterol and fucoxanthin, BACE1 inhibitors from brown algae Undaria pinnatifida and Ecklonia stolonifera. Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer's disease. Arun K.
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Ghosh , Heather L. Rakover , Maria Becker , Beka Solomon. Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores - development of potential agents for the treatment of Alzheimer's disease. References Publications referenced by this paper. Gideon M. Shaked , Markus P. Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes.
These research findings are significant because it will become possible to engineer novel AAV capsids to achieve high-level tissue specific expression. Independent studies have shown that IL transcript and protein levels are reduced in the brains of late onset AD patients as compared to healthy individuals [ 72, 73 ]. These exciting results suggest that targeting neuroinflammation has significant benefits and could potentially lead to development of a multi-target AD gene therapy.
Prior studies by Gray et al. However, when tested in the non-human primates, they observed reduction in peripheral organ and brain transduction with a preferential glial transduction. Their results indicate that high peripheral tropism, limited neuronal transduction in non-human primates, and the pre-existing neutralizing antibodies are the major caveats to human translation of intravascular AAV9 based gene delivery.
Recently, the results from a first-in-human trial of high-dose i. In these trials the patients demonstrated significant improvement in survival and motor function in comparison to the controls. To better understand the efficiency and safety of this approach, Hinderer et al. In yet another study, Hordeaux et al.
They further reported the potential for serious acute toxicity in the non-human primate after systemic administration of high dose of AAV. Therefore, significant caution needs to be exercised during preclinical testing to thoroughly evaluate the safety and toxicity profiles of all the gene therapy vectors in male as well as female subjects. However, one of the major caveats of the current gene therapy strategies is AAV-specific adaptive immune response.
More specifically AAV-neutralizing antibodies will interfere with AAV transduction causing suboptimal transduction rates. Since some of the epitopes are common between different serotypes, it may not be possible to use different serotypes to bypass the immune response. Therefore, it is important to develop suitable strategies to overcome vector directed immune responses. In their pioneering studies, Faust et al.
Recently Hudry et al. The ability of exo-AAV to evade neutralizing antibodies while retaining excellent CNS gene transfer is clinically relevant. One of the major caveats of the current gene therapy vectors is limited CNS gene transfer. CNS gene transfer efficiency and the gene therapy efficacy can be significantly improved by rationally engineering AAV capsid. There have been several studies to maximize the in vivo gene transfer efficiency. Zhong et al. Specific mutation of surface exposed AAV2 capsid tyrosine residues to phenylalanine resulted in improved intracellular transport thereby significantly improving AAV-mediated transduction in vitro as well as in vivo [ 86 ].
Yang et al. Their results suggest that rAAVrh.
Deverman et al. They developed AAV capsid libraries by inserting 7 amino acids of randomized sequence between amino acids and of the AAV9 capsid within the rAAV-Cap-in-cis-lox backbone and delivered the virus libraries to animals with Cre expression in selected cell population. Selective amplification and recovery of sequences that transduced the target population yielded multiple AAV variants. Chan et al. S that efficiently transduced the central and peripheral nervous system, respectively [ 90 ]. Morabito et al. However, whether the selective neurotropism is also preserved in nonhuman primates or human CNS remains to be seen and needs to be investigated in future studies.
In this regard, very recently, Matsuzaki et al. B in marmosets using intravenous injection [ 92 ]. B and AAV9 but marked transduction of the peripheral dorsal root ganglia neurons. Murlidharan et al. They reported the development of a chimeric AAV by replacing the viral protein 3 amino acid residues on AAV9 capsid required for galactose binding on the AAV2 capsid by site directed mutagenesis. The capsid engineered AAV2g9 had a superior CNS transduction efficiency due to preferential neurotropism, CNS restricted biodistribution and minimal transgene expression in off-target peripheral organs.
Tervo et al. It would be interesting to determine whether rAAV-retro vector is capable of efficiently transducing the neural stem cells.
More recently, Choudhury et al. They have developed a novel AAV-B1 capsid, which has superior transduction in the neuronal, glial, and endothelial cell population within the CNS. This AAV-B1 capsid is also highly effective for gene transfer of various peripheral organs including skeletal muscle, heart, lung, pancreas, and retina. They have also generated a novel AAV-AS vector by insertion of a poly-alanine peptide, which imparts 6 and 15 fold more efficient transduction than AAV9 in the spinal cord and cerebrum, respectively [ 96 ].
Kanaan et al. They found that simultaneously mutating multiple tyrosine residues on the AAV2 capsid significantly enhanced neuronal transduction in the striatum and hippocampus and the ablation of heparan sulfate binding also increased the volumetric spread of the vector in vivo.
Clustered regularly interspaced short palindromic repeat CRISPR -Cas nucleases have revolutionized the field of gene editing and have tremendous application in the field of molecular medicine [ 98— ]. We believe that genome editing can significantly improve the development of AD models and also create novel opportunities for the development of the next generation precision targeted AD gene and stem cell therapies. Dahlman et al. Konermann et al.
Using crystallographic studies, they have engineered a combination of sgRNA2. Hu et al. The engineered xCas9 has broadest PAM compatibility and much greater specificity than the regular SpCas9 and substantially lower genome-wide off-target activity. Kramer et al. They discovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, endoplasmic reticulum, proteasome, RNA-processing pathways and chromatin modification.
Their research findings highlight the potential of CRISPR-Cas9 screens in uncovering novel molecular mechanisms underlying neurodegenerative diseases. Paquet et al. Their gene editing approach allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy.
Platt et al. These RosaLSL-Cas9 knock-in mice can be used to generate either single or multiple specific mutations using an AAV vector expressing Cre under the control of a tissue specific promoter along with single or multiple sgRNAs. Zheng et al. They were successful in knocking down the expression of the five genes in the mouse hippocampus using the multiplexing approach. These studies suggest that multiplex CRISPRi can be successfully used to abrogate gene expression in multigeneic neuronal disorders. Prior studies have shown that genetic fusion between dCas9 and the transcriptional repressor KRAB dCas9-KRAB successfully blocks transcription leading to excellent gene knockdown [ — ].
Swiech et al. They observed significant MeCP2 gene editing 7 days post transduction. Their data revealed that triple DNMT knockdown mice had impaired memory formation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy. This has been recently demonstrated by de Solis et al. Surprisingly, their results indicate doxycycline-inducible expression of CRISPR but Tet2 gene editing in a doxycycline independent manner due to leakiness.
This system allowed doxycycline dependent genome editing of Tet2 in N2A cells in vitro. Besides, doxycycline inducible system there are several other inducible systems available including rapamycin, mifepristone, tamoxifen, and ecdysone inducible systems that can be engineered to overcome the leakiness of the dinducible system. The currently used genome editing approaches are inefficient and induce significantly higher random insertions and deletions indels at the target locus. Recently, Nishiyama et al. Using this approach of virus-mediated single-cell labeling of endogenous proteins via HDR vSLENDER , they were able to achieve highly efficient tagging of endogenous proteins in dissociated and organotypic slice cultures in vitro and various brain areas and cell types in developing and adult brains in vivo.
In a pioneering study, Komor et al. These exciting new developments in the field of genome editing have the potential to revolutionize the development of next generation patient-specific precision medicine for a wide variety of genetic diseases as well as neurodegenerative diseases especially AD. Bustos et al. B vectors expressing artificial transcription factors or epigenetic factors methylating H3K9.
These exciting results suggest that epigenetic editing could be utilized for developing novel gene therapies for the treatment of AD. Development of novel AD therapies would involve harnessing the latest technological advancements in the fields of neurology, immunology, molecular biology, virology, molecular medicine as well as stem cell biology. Neuroinflammation plays a significant role in the development and progression of AD. Genetically engineered recombinant viral vectors with enhanced neurotropism due to novel capsid engineering will maximize targeted gene editing and gene therapy efficacy.
Latest advancements in the field of stem cell biology and regenerative medicine will enable the development of disease in a dish model using AD patient derived iPS cells to generate 3D organoids, which can be used for the new drug discovery. Such an approach will lead to the development of AD-patient specific neuro-immuno-genome-editing-stem-cell-therapy. In a large scale genome-wide survival analysis of 14, AD cases and 25, controls Huang et al. They discovered an association with delayed AD onset and lower expression of SPl1 in monocytes and macrophages.
The SPl1 gene encodes for the PU. There is evidence linking AD related pathology with increased hippocampal neurogenesis in AD patients. Jin et al. They found that the expression of several neurogenesis markers was increased in the hippocampus of severely affected AD patients.
These findings are in contrast to the findings in AD transgenic mice, which show impaired hippocampal neurogenesis [ — ]. Baglietto-Vargas et al. There is a need to investigate the role of hippocampal neurogenesis in AD pathology as well as cognitive function. AD in a dish model for precision medicine. AD patient-specific fibroblasts can be reprogrammed into iPS cells. Progressive stepwise differentiation and lineage commitment of AD patient-specific iPS cells will generate iNeurons, iAstrocytes, and iMicroglia which play a crucial role in AD pathogenesis.
Advancements in biomedical engineering have made it possible to generate 3D brain-like mini organoids derived from iPS cells, which can be very valuable in robotic high throughput screening of novel AD therapeutic targets. Once a lead compound with significant therapeutic potential is identified it can be used for the dose optimization, preclinical, pharmacokinetic, pharmacodynamic, toxicology, and safety studies in AD murine and non-human primate models.
Finally, utilizing crystallography, mass-spectrometry, bioinformatics tools and latest advancements in precision medicine, AD patient-specific novel therapies can be successfully developed. Han et al. They created a human ScFv lentiviral intracellular combinatorial antibody library with 10 8 unique antibody clones. Total bone marrow cells were harvested from mice, transduced with the lentiviral library and then transplanted into lethally irradiated mice. Seven days post transplantation, various organs were harvested and the cells assessed for migration and the DNA was subjected to PCR to amplify and sequence human ScFv sequences.
They were able to identify an antibody B1 that induced migration of the cells to the brain. These exciting results could potentially lead to the development of novel therapeutic strategies to successfully treat AD. There has been a significant interest in developing novel AD therapies. However, despite significant global efforts, successful AD therapy remains elusive. There are multiple hurdles and roadblocks that need to be overcome before a precision-targeted patient-specific AD therapy becomes a reality.
Toward our commitment to our long-term goal of developing targeted gene-editing and stem cell-based patient-specific therapies against AD and diabetes, we have very recently proposed a novel paradigm wherein tanycytes have been implicated to play a crucial role in diabetes and AD pathogenesis [ ].
Further, we believe that there is critical need to focus our efforts on the deciphering the nexus between traumatic brain injury, post-traumatic stress disorder and AD as it will enable us to uncover potentially novel molecular mechanisms underlying AD initiation and progression [ , ]. Science , — J Clin Invest , — Neurobiol Dis 14, — Neurobiol Dis 23, — J Alzheimers Dis 6, — J Neurosci 26, — Mol Ther 16, — Mol Ther 17, — Neuropsychopharmacology 39, — J Exp Med , — J Neurochem , — Even if the initial intent of these modifications is reparative, such long-lasting and uncontrolled activation causes further neurodegeneration.
Even astrocytes play an important role in the maintenance of the cerebral homeostasis. In physiological conditions, microglia protects the brain from pathogens, and, together with macroglia, helps maintain homeostasis of the tissue. In AD, all these cells became more reactive and change their morphology surrounding SPs.
To the vicious circle driven by cytokines and MAPKs, 83 , 84 the resulting activation of the complement cascade has to be added, 85 as well as the induction of proinflammatory enzymes, such as cyclooxygenase-2 COX-2 86 and the inducible nitric oxide synthase iNOS. These proteins, produced by activated microglia and macrophages, are increased in AD brain and are responsible for protein complex formation. Studies report that tau hyperphosphorylation is directly affected by inflammatory mediators, including the cyclin-dependent kinase 5 CDK5 : 93 IL-6 stimulates neuronal protein p35, which in turn is responsible for the kinase activation that can act on tau.
Recently, the role of protein kinase 2 CK2, former casein kinase II has been described.
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In fact, CK2 immunopositive astrocytes have been found to be associated with amyloid deposits in AD brains, suggesting its involvement in the neuroinflammatory response. Inflammatory mediators, in particular cytokines, are also responsible for increased BBB permeabilization driven by chemokines, allowing leukocyte penetration in the brain. The salient events reported in this paragraph are summarized in Figure 2. Figure 2 Integrated pathways between glial activation, neuroinflammation, and neuronal death after brain injury.
Notes: Whenever a brain injury occurs, glial activation takes place with the aim of removing injurious stimuli. To this aim, activated cells undergo a series of morphofunctional changes and acquire a reactive phenotype. Activation causes, among other things, glial hypertrophy, astrocyte endfeet retraction, and gain of amoeboid microglial structure.
These changes, if not stopped, can induce synaptic dysfunction, homeostatic imbalance, neurovascular unit dysfunction, loss of three-dimensional network, and BBB dysfunction.
Development and Structural Modification of BACE1 Inhibitors
In addition, reactive microglia and astrocytes release a wide range of proinflammatory mediators aimed at removing the primary injury. The occurrence of a reactive state is very probably a protective response. However, uncontrolled and prolonged activation goes beyond physiological control, and detrimental effects override the beneficial ones. Solid arrows indicate complete pathways. Dashed arrows indicate pathways that that occur partially or do not. Because of the knowledge acquired so far and the failure of so many antiamyloid trials, scientific interest has shifted to other features of neurodegeneration including neuroinflammation.
Recently, much work has been done, but much more research still needs to be done. It is now clear that the AD neurodegenerative process is also orchestrated by proinflammatory cytokines and their receptors, which therefore become promising targets on which to focus by means of different approaches. Blocking gene expression of cytokines, releasing or binding their receptors, or better regulating the functioning of cells implicated in the neuroinflammation are definitely strategies still in exploration.
Interestingly, both in vitro and in vivo studies have shown that pharmacological inhibition of COX-2 and inducible NO synthase has positive outcomes. Lastly, in AD models it was observed that it is possible to obtain satisfactory results by modulating kinases that are not only directly related to tau hyperphosphorylation but also to neuroinflammation. Experimental studies have shown that it is possible to exert anti-inflammatory effect by inhibiting this enzyme, giving us another potential therapeutic target to consider.
Currently available products and products in research and development focusing on neuroinflammatory targets. In the past decades, several epidemiological and clinical studies were carried out to demonstrate the neuroprotective potential of several nonsteroidal anti-inflammatory drugs. Once again, results were disappointing. For these reasons, an ideal anti-inflammatory compound should be able to control the detrimental effects and, at the same time, preserve the physiological glial activation.
An alternative and recent therapeutic approach is represented by nutraceuticals eg, curcumin, apigenin, docosahexaenoic acid, resveratrol, and n-3 fatty acids. A large number of studies have been done in this field, and promising data were obtained in preclinical models. Unfortunately, these encouraging findings were not replicated in clinical trials, and promising vaccines were stopped because of adverse effects such as meningoencephalitis.
Presently, several competing hypotheses especially related to time of intervention may help explain the failure of translating preclinical studies into the clinical ones, but so far there is no way to confirm which of these explanations is correct. The pathogenic role of neuroinflammation in AD is now well recognized and accepted. Nevertheless, the underlying mechanisms have not been sufficiently elucidated. Several factors contribute to this failure.
First of all, there is a lack of adequate preclinical models that best mimic the disease and, in particular, the processes of glial activation and neuroinflammation. Then, another important factor is the comprehension of the role of each cellular component in the inflammatory process, for example, the identification of cell-specific biomarkers. Finally, it is important to define the inflammatory stages to correlate each phase to AD progression and to clarify which processes are protective and which ones are detrimental. The achievement of these goals will allow scientists to practice many other experimental approaches.
The resulting production of cytokines and proinflammatory molecules has initially a neuroprotective role, but subsequently becomes the cause of further neurodegeneration. Unfortunately, because of the lack of appropriate animal models, we still lack a complete understanding of the relationship between inflammatory process stages and AD progression. This could explain, at least in part, the unsuccessful results of clinical trials performed with anti-inflammatory molecules whose efficacy was significantly proven in preclinical investigations.
Therefore, future experimental studies must intensively investigate the intricate paths of the neuroinflammatory process and define the best time to control it. In this way, it will be possible to achieve more focused and functional therapeutic strategies in the hope of not only alleviating but also modifying AD progression.
The authors thank Miss Federica Bellifemine for her helpful contribution in preparing the figure 1. Imaging of neuroinflammation in dementia: a review. J Neurol Neurosurg Psychiatry. World Alzheimer Report Alzheimer A. Uber eine eigenartige Erkangkung der Hirnrinde An unusual illness of the cerebral cortex.
Neuro-Immuno-Gene- and Genome-Editing-Therapy for Alzheimer’s Disease: Are We There Yet?
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