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Parkinson’s disease is characterized by the gradual appearance of intraneuronal inclusions that are primarily composed of misfolded α-synuclein protein, leading to cytotoxicity and neural death. Recent in vitro and in vivo studies suggest that misfolded α-synuclein may spread transcellularly in a prion-like manner, inducing pathological aggregates in healthy neurons, and is disseminated via secretion of extracellular vesicles. Accordingly, extracellular vesicles derived from brain lysates and CSF of patients with Parkinson’s disease were shown to facilitate α-synuclein aggregation in healthy cells. Prompted by the hypothesis of Braak and colleagues that the olfactory bulb is one of the primary propagation sites for the initiation of Parkinson’s disease, we sought to investigate the role of extracellular vesicles in the spread of α-synuclein and progression of Parkinson’s disease through the olfactory bulb. Extracellular vesicles derived from the CSF of patients diagnosed with Parkinson’s disease or with a non-synucleinopathy neurodegenerative disorder were administered intranasally to healthy mice, once daily over 4 days. Three months later, mice were subjected to motor and non-motor tests. Functional impairments were elucidated by histochemical analysis of midbrain structures relevant to Parkinson’s disease pathology, 8 months after EVs treatment. Mice treated with extracellular vesicles from the patients with Parkinson’s disease displayed multiple symptoms consistent with prodromal and clinical-phase Parkinson’s disease such as hyposmia, motor behaviour impairments and high anxiety levels. Furthermore, their midbrains showed widespread α-synuclein aggregations, dopaminergic neurodegeneration, neuroinflammation and altered autophagy activity. Several unconventional pathologies were also observed, such as α-synuclein aggregations in the red nucleus, growth of premature grey hair and astrogliosis. Collectively, these data indicate that intranasally administered extracellular vesicles derived from the CSF of patients with Parkinson’s disease can propagate α-synuclein aggregation in vivo and trigger Parkinson’s disease-like symptoms and pathology in healthy mice.

The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. As wildtype SpyCas9 is known to generate many off-target effects, its use in the clinic remains an elusive goal. To date, several high-fidelity Cas9 variants with improved specificity over wildtype SpyCas9 have been described. Each of the variants was engineered either by rational design or experimental directed evolution. However, the potential of improved specificity using both methods is constrained to specific residues due to the requirement of a priori knowledge and challenges in selecting engineered proteins, which arise from factors such as selection pressure, assay limitations, host organism compatibility, and library size and diversity. Therefore, SpyCas9 modifications through in-silico protein engineering allow an efficient specificity improvement while overcoming those limitations. Nevertheless, the lack of living models to test the evolved protein remains a major challenge of computational protein engineering methods. We recently demonstrated the advantage of normal mode analysis to simulate and predict the enzymatic function of SpyCas9 in the presence of mismatches. Here, we report ComPE, a novel computational protein engineering method to modify the protein and measure the vibrational entropy of wildtype or variant SpyCas9 in complex with its sgRNA and target DNA. Using this platform, we discovered novel high-fidelity Cas9 variants with improved specificity. We functionally validated the improved specificity of four variants, and the intact on-target activity in one of them. Lastly, we demonstrate their reduced off-target editing and non-specific gRNA-independent DNA damage, highlighting their advantages for clinical applications. The described method could be applied to a wide range of proteins, from CRISPR-Cas orthologs to distinct proteins in any field where engineered proteins can improve biological processes.

The CRISPR-Cas system has transformed the field of gene-editing and created opportunities for novel genome engineering therapeutics. The field has significantly progressed, and recently, CRISPR-Cas9 was utilized in clinical trials to target disease-causing mutations. Existing tools aim to predict the on-target efficacy and potential genome-wide off-targets by scoring a particular gRNA according to an array of gRNA design principles or machine learning algorithms based on empirical results of large numbers of gRNAs. However, such tools are unable to predict the editing outcome by variant Cas enzymes and can only assess potential off-targets related to reference genomes. Here, we employ normal mode analysis (NMA) to investigate the structure of the Cas9 protein complexed with its gRNA and target DNA and explore the function of the protein. Our results demonstrate the feasibility and validity of NMA to predict the activity and specificity of SpyCas9 in the presence of mismatches by comparison to empirical data. Furthermore, despite the absence of their exact structures, this method accurately predicts the enzymatic activity of known high-fidelity engineered Cas9 variants.

Neurological disorders are diseases of the central nervous system (CNS), characterized by a progressive degeneration of cells and deficiencies in neural functions. Mesenchymal stem cells (MSCs) are a promising therapy for diseases and disorders of the CNS. Increasing evidence suggests that their beneficial abilities can be attributed to their paracrine secretion of extracellular vesicles (EVs). Administration of EVs that contain a mixture of proteins, lipids, and nucleic acids, resembling the secretome of MSCs, has been shown to mimic most of the effects of the parental cells. Moreover, the small size and safety profile of EVs provide a number of advantages over cell transplantation. Intranasal (IN) administration of EVs has been established as an effective and reliable way to bypass the blood-brain barrier and deliver drugs to the CNS. In addition to pharmacological drugs, EVs can be loaded with a diverse range of cargo designed to modulate gene expression and protein functions in recipient cells, and lead to immunomodulation, neurogenesis, neuroprotection, and degradation of protein aggregates. In this review, we will explore the proposed physiological pathways by which EVs migrate through the nasal route to the CNS where they can actively target a region of injury or inflammation and exert their therapeutic effects. We will summarize the functional outcomes observed in animal models of neurological diseases following IN treatment with MSC-derived EVs. We will also examine key mechanisms that have been suggested to mediate the beneficial effects of EV-based therapy.

The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients.

Autism spectrum disorder (ASD) is a neurodevelopmental disease with a wide spectrum of manifestation. The core symptoms of ASD are persistent deficits in social communication, and restricted and repetitive patterns of behavior, interests, or activities. These are often accompanied by intellectual disabilities. At present, there is no designated effective treatment for the core symptoms and co-morbidities of ASD. Recently, interest is rising in medical cannabis as a treatment for ASD, with promising clinical data. However, there is a notable absence of basic pre-clinical research in this field. In this study, we investigate the behavioral and biochemical effects of long-term oral treatment with CBD-enriched medical cannabis oil in a human mutation-based Shank3 mouse model of ASD. Our findings show that this treatment alleviates anxiety and decreases repetitive grooming behavior by over 70% in treated mutant mice compared to non-treated mutant mice. Furthermore, we were able to uncover the involvement of CB1 receptor (CB1R) signaling in the Avidekel oil mechanism, alongside a mitigation of cerebrospinal fluid (CSF) glutamate concentrations. Subsequently, RNA sequencing (RNA seq) of cerebellar brain samples revealed changes in mRNA expression of several neurotransmission-related genes post-treatment. Finally, our results question the relevancy of CBD enrichment of medical cannabis for treating the core symptoms of ASD, and emphasize the importance of the THC component for alleviating deficits in repetitive and social behaviors in ASD.

Tissue-specific uptake and sufficient biodistribution are central goals in drug development. Crossing the blood-brain barrier (BBB) represents a major challenge in delivering therapeutics to the central nervous system (CNS). Since its discovery in the late 19th century, considerable efforts have been invested in an attempt to decipher the BBB structure complexity and plasticity. In parallel, another prevalent approach is to improve a delivery system by harnessing the biological machinery in an attempt to enhance therapeutic-agent permeability. Here, we review the advantages and limitations of using extracellular vesicles over AAV systems as a delivery system for therapy, focusing on neurodevelopmental disorders.

Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function and, most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.

Background: Alzheimer's Disease (AD) is associated with impairments in key brain Mitogen- Activated Protein Kinase (MAPK) signaling cascades including the p38, c-Jun N-terminal kinase (JNK), ERK and Akt pathways. Apolipoprotein E4 (ApoE4) is the most prevalent genetic risk factor of AD. Objectives: To investigate the extent to which the MAPK signaling pathway plays a role in mediating the pathological effects of apoE4 and can be reversed by experimental manipulations. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Results: ApoE4 mice showed robust activation of the stress related p38 and JNK pathways and a corresponding decrease in Akt activity, which is coupled to activation of GSK3β and tau hyperphosphorylation. There was no effect on the ERK pathway. We have previously shown that the apoE4- related pathology, namely; accumulation of Aβ, hyper-phosphorylated tau, synaptic impairments and decreased VEGF levels can be reversed by up-regulation of VEGF level utilizing a VEGF-expressing adeno-associated virus. Utilizing this approach, we assessed the extent to which the AD-hallmark and synaptic pathologies of apoE4 are related to the corresponding MAPK signaling effects. This revealed that the reversal of the apoE4-driven pathology via VEGF treatment was associated with a reversal of the p38 and Akt related effects. Conclusion: Taken together, these results suggest that the p38 and Akt pathways play a role in mediating the AD-related pathological effects of apoE4 in the hippocampus.

Exosomes, nanovesicles that are secreted by different cell types, enable intercellular communication at local or distant sites. Alhough they have been found to cross the blood brain barrier, their migration and homing abilities within the brain remain unstudied. We have recently developed a method for longitudinal and quantitative in vivo neuroimaging of exosomes based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with gold nanoparticles as labeling agents. Here, we used this technique to track the migration and homing patterns of intranasally administrated exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) in different brain pathologies, including stroke, autism, Parkinson's disease, and Alzheimer's disease. We found that MSC-exo specifically targeted and accumulated in pathologically relevant murine models brains regions up to 96 h post administration, while in healthy controls they showed a diffuse migration pattern and clearance by 24 h. The neuro-inflammatory signal in pathological brains was highly correlated with MSC-exo accumulation, suggesting that the homing mechanism is inflammatory-driven. In addition, MSC-exo were selectively uptaken by neuronal cells, but not glial cells, in the pathological regions. Taken together, these findings can significantly promote the application of exosomes for therapy and targeted drug delivery in various brain pathologies.

Neuronal membranes contain exceptionally high concentrations of long-chain polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), which are essential for neuronal development and function. Adult bone-marrow-derived mesenchymal stem cells (MSC) can be induced to possess some neuronal characteristics. Here we examined the effects of neuronal induction on the PUFA metabolism specific pathways. Differentiated cells contained ~30% less ARA than MSC. The expression of specific ARA metabolizing enzymes was upregulated, notably that of prostaglandin E2 synthase which increased more than 15-fold, concomitantly with a 3-fold increase in the concentration of PGE2 in the medium. Moreover, induced differentiation was associated with enhanced incorporation of exogenous DHA, upregulation of acyl-CoA synthases, fatty acid binding proteins, choline kinase (CK) and phosphatidylserine synthases as well as increased total cellular phospholipids (PL). These findings suggest that active ARA metabolites may be important in the differentiation process and that neuronal induction prepares the resulting cells for increased DHA incorporation through the action of specific enzymes.

This chapter describes animal models for autism, with a focus on RRBIs. ASD evidently has a robust genetic component, with strong familial inheritance patterns and the potential involvement of approximately 1000 genes. Research supports that RRBIs have a strong genetic component, and there is some evidence that an imbalance in the neurotransmission system of dopamine, GABA, and serotonin (5-HT) have a role in RRBI. The chapter describes animal models for autism, with a focus on RRBI. Findings from three different mouse models are presented: a mouse model based on mutations found in humans with ASD (the mouse genome is altered to include the same mutations as those found in humans with ASD), a mouse model of ASD involving neurodevelopmental processes during pregnancy, and a multifactorial mouse model that includes a combination of factors other than genetic mutations or biological markers that account for the observed ASD-related symptoms. The studies on these mouse models, similar to the research on neurological mechanisms among humans, emphasize our current understanding of ASD in humans. That is, some ASD symptoms may or may not be associated with various genetic and environmental factors, and the etiology of ASD and RRBI in most individuals with these diagnoses remains an enigma.

The etiology of major neurodevelopmental disorders such as schizophrenia and autism is unclear, with evidence supporting a combination of genetic factors and environmental insults, including viral infection during pregnancy. Here we utilized a mouse model of maternal immune activation (MIA) with the viral mimic PolyI:C infection during early gestation. We investigated the transcriptional changes in the brains of mouse fetuses following MIA during the prenatal period, and evaluated the behavioral and biochemical changes in the adult brain. The results reveal an increase in RNA editing levels and dysregulation in brain development-related gene pathways in the fetal brains of MIA mice. These MIA-induced brain editing changes are not observed in adulthood, although MIA-induced behavioral deficits are observed. Taken together, our findings suggest that MIA induces transient dysregulation of RNA editing at a critical time in brain development., Interferon can be induced by the viral mimic PolyI:C, which can in turn activate RNA editing enzymes. Here the authors identify transient changes in A-to-I RNA editing following maternal immune activation in mice.

Schizophrenia is a debilitating psychiatric disorder with a significant number of patients not adequately responding to treatment. Phencyclidine (PCP) is used as a validated model for schizophrenia, shown to reliably induce positive, negative and cognitive-like behaviors in rodents. It was previously shown in our lab that behavioral phenotypes of PCP-treated mice can be alleviated after intracranial transplantation of mesenchymal stem cells (MSC). Here, we assessed the feasibility of intranasal delivery of MSCs-derived-extracellular vesicles (EVs) to alleviate schizophrenia-like behaviors in a PCP model of schizophrenia. As MSCs-derived EVs were already shown to concentrate at the site of lesion in the brain, we determined that in PCP induced injury the EVs migrate to the prefrontal cortex (PFC) of treated mice, a most involved area of the brain in schizophrenia. We show that intranasal delivery of MSC-EVs improve social interaction and disruption in prepulse inhibition (PPI) seen in PCP-treated mice. In addition, immunohistochemical studies demonstrate that the EVs preserve the number of parvalbumin-positive GABAergic interneurons in the PFC of treated mice. Finally, MSCs-EVs reduced glutamate levels in the CSF of PCP-treated mice, which might explain the reduction of toxicity. In conclusion, we show that MSCs-EVs improve the core schizophrenia-like behavior and biochemical markers of schizophrenia and might be used as a novel treatment for this incurable disorder.

Schizophrenia is a debilitating psychiatric disorder with a significant number of patients not adequately responding to treatment. Phencyclidine (PCP) is used as a validated model for schizophrenia, shown to reliably induce positive, negative and cognitive-like behaviors in rodents. It was previously shown in our lab that behavioral phenotypes of PCP-treated mice can be alleviated after intracranial transplantation of mesenchymal stem cells (MSC). Here, we assessed the feasibility of intranasal delivery of MSCs-derived-extracellular vesicles (EVs) to alleviate schizophrenia-like behaviors in a PCP model of schizophrenia. As MSCs-derived EVs were already shown to concentrate at the site of lesion in the brain, we determined that in PCP induced injury the EVs migrate to the prefrontal cortex (PFC) of treated mice, a most involved area of the brain in schizophrenia. We show that intranasal delivery of MSC-EVs improve social interaction and disruption in prepulse inhibition (PPI) seen in PCP-treated mice. In addition, immunohistochemical studies demonstrate that the EVs preserve the number of parvalbumin-positive GABAergic interneurons in the PFC of treated mice. Finally, MSCs-EVs reduced glutamate levels in the CSF of PCP-treated mice, which might explain the reduction of toxicity. In conclusion, we show that MSCs-EVs improve the core schizophrenia-like behavior and biochemical markers of schizophrenia and might be used as a novel treatment for this incurable disorder.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising novel technology that holds the potential of treating genetic diseases. Safety and specificity of the treatment are to be further studied and developed prior to implementation of the technology into the clinic. The guide-RNA (gRNA) allows precise position-specific DNA targeting, although it may tolerate small changes such as point mutations. The permissive nature of the CRISPR-Cas system makes allele-specific targeting a challenging goal. Hence, an allele-specific targeting approach is in need for future treatments of heterozygous patients suffering from diseases caused by dominant negative mutations. The single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach allows highly allele-specific DNA cleavage due to the existence of a novel PAM sequence only at the target allele. Here, we present CrisPam, a computational tool that detects PAMs within the variant allele for allele-specific targeting by CRISPR-Cas systems. The algorithm scans the sequences and attempts to identify the generation of multiple PAMs for a given reference sequence and its variations. A successful result is such that at least a single PAM is generated by the variation nucleotide. Since the PAM is present within the variant allele only, the Cas enzyme will bind the variant allele exclusively. Analyzing a dataset of human pathogenic point mutations revealed that 90% of the analyzed mutations generated at least a single PAM. Thus, the SNP-derived PAM approach is ideal for targeting most of the point mutations in an allele-specific manner. CrisPam simplifies the gRNAs design process to specifically target the allele of interest and scans a wide range of 26 unique PAMs derived from 23 Cas enzymes. CrisPam is freely available at https://www.danioffenlab.com/crispam.

Background Partial or an entire deletion of SHANK3 are considered as major drivers in the Phelan–McDermid syndrome, in which 75% of patients are diagnosed with autism spectrum disorder (ASD). During the recent years, there was an increasing interest in stem cell therapy in ASD, and specifically, mesenchymal stem cells (MSC). Moreover, it has been suggested that the therapeutic effect of the MSC is mediated mainly via the secretion of small extracellular vesicle that contains important molecular information of the cell and are used for cell-to-cell communication. Within the fraction of the extracellular vesicles, exosomes were highlighted as the most effective ones to convey the therapeutic effect. Methods Exosomes derived from MSC (MSC-exo) were purified, characterized, and given via intranasal administration to Shank3B KO mice (in the concentration of 107 particles/ml). Three weeks post treatment, the mice were tested for behavioral scoring, and their results were compared with saline-treated control and their wild-type littermates. Results Intranasal treatment with MSC-exo improves the social behavior deficit in multiple paradigms, increases vocalization, and reduces repetitive behaviors. We also observed an increase of GABARB1 in the prefrontal cortex. Conclusions Herein, we hypothesized that MSC-exo would have a direct beneficial effect on the behavioral autistic-like phenotype of the genetically modified Shank3B KO mouse model of autism. Taken together, our data indicate that intranasal treatment with MSC-exo improves the core ASD-like deficits of this mouse model of autism and therefore has the potential to treat ASD patients carrying the Shank3 mutation.

One of the pathways of the unfolded protein response, initiated by PKR-like endoplasmic reticulum kinase (PERK), is key to neuronal homeostasis in neurodegenerative diseases. PERK pathway activation is usually accomplished by inhibiting eIF2α-P dephosphorylation, after its phosphorylation by PERK. Less tried is an approach involving direct PERK activation without compromising long-term recovery of eIF2α function by dephosphorylation. Here we show major improvement in cellular (STHdhQ111/111) and mouse (R6/2) Huntington's disease (HD) models using a potent small molecule PERK activator that we developed, MK-28. MK-28 showed PERK selectivity in vitro on a 391-kinase panel and rescued cells (but not PERK−/− cells) from ER stress-induced apoptosis. Cells were also rescued by the commercial PERK activator CCT020312 but MK-28 was significantly more potent. Computational docking suggested MK-28 interaction with the PERK activation loop. MK-28 exhibited remarkable pharmacokinetic properties and high BBB penetration in mice. Transient subcutaneous delivery of MK-28 significantly improved motor and executive functions and delayed death onset in R6/2 mice, showing no toxicity. Therefore, PERK activation can treat a most aggressive HD model, suggesting a possible approach for HD therapy and worth exploring for other neurodegenerative disorders.

Background: Partial or an entire deletion of SHANK3 are considered as major drivers in the Phelan McDermid syndrome, in which 75% of patients are diagnosed with autism spectrum disorder (ASD). During the recent years, there was an increasing interest in stem cell therapy in ASD, and specifically, mesenchymal stem cells (MSC). Moreover, it has been suggested that the therapeutic effect of the MSC is mediated mainly via the secretion of small extracellular-vesicle that contain important molecular information of the cell and are used for cell-to cell communication. Within the fraction of the extracellular-vesicles, exosomes were highlighted as the most effective ones to convey the therapeutic effect.Methods: Exosomes derived from MSC (MSC-exo) were purified, characterized and given via intranasal administration to Shank3B KO mice (in concentration of 107 particals/ml). Three weeks post treatment the mice were tested for behavioral scoring and their results were compared to control saline treated and to their wild type littermates. Results: Intranasal treatment with MSC-exo improves the social behavior deficit in multiple paradigms, increases vocalization and reduces repetitive behaviors. We also observed an increase of GABARB1 in the prefrontal cortex.Conclusions: Herein, we hypothesized that MSC-exo would have a direct beneficial effect on the behavioral autistic-like phenotype of the genetically modified Shank3B KO mice model of autism. Taken together, our data indicate that intranasal treatment with MSC-exo improves the core ASD-like deficits of this mouse model autism and therefore has the potential to treat ASD patients carrying the Shank3 mutation.Limitations: Although the behavioral scoring of the MSC-exo treated group was significantly improved compared to their saline treated littermates, there is much more to learn regarding the mechanism of action of the MSC-exo.

Exosomes, a subtype of extracellular vesicles, are nanovesicles of endocytic origin. Exosomes contain a plethora of proteins, lipids, and genetic materials of parent cells to facilitate intercellular communications. Tracking exosomes in vivo is fundamentally important to understand their biodistribution pattern and the mechanism of biological actions in experimental models. Until now, a number of tracking protocols have been developed, including fluorescence labeling, bioluminescence imaging, magnetic resonance imaging, and computed tomography (CT) tracking of exosomes. Recently, we have shown the tracking and quantification of exosomes in a spinal cord injury model, by using two tracking approaches. More specifically, following intranasal administration of gold nanoparticle-encapsulated exosomes to rats bearing complete spinal cord injury, exosomes in the whole central nervous system were tracked by using microCT, and quantified by using inductively coupled plasma and flame atomic absorption spectroscopy. In addition, optical imaging of fluorescently labeled exosomes was performed to understand the abundance of migrating exosomes in the spinal cord lesion, as compared to the healthy controls, and to further examine their affinity to different cell types in the lesion. Thus, the protocol presented here aids in the study of exosome biodistribution at both cellular and organ levels, in the context of spinal cord injury. This protocol will also enable researchers to better elucidate the fate of administered exosomes in other models of interest.

Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available athttps://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

Research into stroke is driven by frustration over the limited available therapeutics. Targeting a single aspect of this multifactorial disease contributes to the therapeutic boundaries. To overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding excitatory amino acid transporter 2 (EAAT2(, glutamate dehydrogenase 2 (GDH2), and nuclear factor E2-related factor 2 (Nrf2) genes, that acts synergistically to address the effected excito-oxidative axis. Here, we used the vasoconstrictor endothelin-1 (ET-1) to induce focal ischemic injury in mice by direct injection into the striatum. Mice treated with the mixture of these three genes show significant improvement in body balance, motor coordination, and decreased motor asymmetry compared to each gene separately. These results demonstrate that overexpression of the combined EAAT2, GDH2, and NRF2 genes can provide neuroprotection after ischemic injury.

Autism spectrum disorders (ASD) are neurodevelopmental disabilities characterized by severe impairment in social communication skills and restricted, repetitive behaviors. We have previously shown that a single transplantation of mesenchymal stem cells (MSC) into the cerebral lateral ventricles of BTBR autistic-like mice resulted in an improvement across all diagnostic criteria of ASD. We suggested that brain-derived neurotrophic factor (BDNF), a protein which supports the survival and regeneration of neurons secreted by MSC, largely contributed to the beneficial behavioral effect. In this study, we investigated the behavioral effects of transplanted MSC induced to secrete higher amounts of neurotrophic factors (NurOwn®), on various ASD-related behavioral domains using the BTBR mouse model of ASD. We demonstrate that NurOwn® transplantation had significant advantages over MSC transplantation in terms of improving communication skills, one and six months following treatment, as compared to sham-treated BTBR mice. Furthermore, NurOwn® transplantation resulted in reduced stereotypic behavior for as long as six months post treatment, compared to the one month improvement observed in the MSC treated mice. Notably, NurOwn® treatment resulted in improved cognitive flexibility, an improvement that was not observed by MSC treatment. Both MSC and NurOwn® transplantation induced an improvement in social behavior that lasted for six months. In conclusion, the present study demonstrates that a single transplantation of MSC or NurOwn® have long-lasting benefits, while NurOwn® may be superior to MSC treatment.

Physical exercise has gained increasing interest as a treatment modality that improves prognosis in psychiatric patients. The disrupted in schizophrenia 1 (DISC1) gene is a candidate gene for major mental illness. In this study, we aimed to determine whether voluntary wheel running can improve cognitive deficits of dominant-negative DISC1 transgenic mice (DN-DISC1).DN-DISC1 and control mice (10-week-old male and female) were placed for 14 days in a cage with or without access to a running wheel. Two weeks later, mice underwent behavioural tests evaluating cognition and social approach and recognition.Voluntary exercise improved performance in the novel object recognition test, restored the impairment in spatial memory in the Y maze, and reversed the deficit in social recognition memory in DN-DISC1 females. DN-DISC1 males did not exhibit behavioural deficits at baseline. Tissue analysis revealed that exercise induced a significant increase in hippocampal expression of doublecortin (DCX), brain-derived neurotrophic factor (BDNF) and cannabinoid receptor type 1 (CB1R) only in DN-DISC1 females.Voluntary exercise is beneficial in attenuating cognitive deficits observed in a rodent model relevant for neuropsychiatric disorders. The data add a preclinical aspect to the accumulating clinical data supporting the incorporation of physical exercise to patients' care.

Cannabis abuse in adolescence is associated with increased risk of psychotic disorders. Δ-9-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. Disrupted-In-Schizophrenia-1 (DISC1) protein is a driver for major mental illness by influencing neurodevelopmental processes. Here, utilizing a unique mouse model based on host (DISC1) X environment (THC administration) interaction, we aimed at studying the pathobiological basis through which THC exposure elicits psychiatric manifestations. Wild-Type and dominant-negative-DISC1 (DN-DISC1) mice were injected with THC (10 mg/kg) or vehicle for 10 days during mid-adolescence-equivalent period. Behavioral tests were conducted to assess exploratory activity (open field test, light-dark box test) and cognitive function (novel object recognition test). Electrophysiological effect of THC was evaluated using acute hippocampal slices, and hippocampal cannabinoid receptor type 1 and brain-derived neurotrophic factor (BDNF) protein levels were measured. Our results indicate that THC exposure elicits deficits in exploratory activity and recognition memory, together with reduced short-term synaptic facilitation and loss of BDNF surge in the hippocampus of DN-DISC mice, but not in wild-type mice. Over-expression of BDNF in the hippocampus of THC-treated DN-DISC1 mice prevented the impairment in recognition memory. The results of this study imply that induction of BDNF following adolescence THC exposure may serve as a homeostatic response geared to maintain proper cognitive function against exogenous insult. The BDNF surge in response to THC is perturbed in the presence of mutant DISC1, suggesting DISC1 may be a useful probe to identify biological cascades involved in the neurochemical, electrophysiological, and behavioral effects of cannabis related psychiatric manifestations.

SummaryObjective Epilepsy affects 60 million people worldwide. Despite the development of antiepileptic drugs, up to 35% of patients are drug refractory with uncontrollable seizures. Toll-like receptors (TLRs) are central components of the nonspecific innate inflammatory response. Because TLR3 was recently implicated in neuronal plasticity, we hypothesized that it may contribute to the development of epilepsy after status epilepticus (SE). Methods To test the involvement of TLR3 in epileptogenesis, we used the pilocarpine model for SE in TLR3-deficient mice and their respective wild-type controls. In this model, a single SE event leads to spontaneous recurrent seizures (SRS). Two weeks after SE, mice were implanted with wireless electroencephalography (EEG) transmitters for up to 1 month. The impact of TLR3 deficiency on SE was assessed using separate cohorts of mice regarding EEG activity, seizure progression, hippocampal microglial distribution, and expression of the proinflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)β. Results Our data indicate that TLR3 deficiency reduced SRS, microglial activation, and the levels of the proinflammatory cytokines TNFα and IFNβ, and increased survival following SE. Significance This study reveals novel insights into the pathophysiology of epilepsy and the contribution of TLR3 to disease progression. Our results identify the TLR3 pathway as a potential future therapeutic target in SE.

Background & Aim Background: During the recent decade, exosomes that derived from mesenchymal stem cells (MSC-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. We have previously shown that intranasal administration of MSC-exo, cross the BBB and significantly ameliorate autistic like behavioral phenotype in BTBR and SHANK3 animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (ASD). Objective: In order to study the mechanism of action and the cellular pathways in which the adipose-derived MSC-exo activate their target, we preformed RNA sequencing analysis of primary neurons isolated from SHANK3 mice treated with MSC-exo. Methods, Results & Conclusion Methods: Primary neuronal cell cultures were prepared from newborn SHANK3 homozygotes mice model of autism. Cultures were treated with 40ul MSC-exo (107 particles/ul), isolated from human adipocytes, followed by RNA sequencing. The alterations in gene expression between the treated and intact neurons were analyzed for gene ontology and pathways and were also compared to proteomics analysis of the MSC-exo in order to find regulatory proteins that may lead to these differences. Results: Bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with MSC-exo. One of them is BDNF which is known as an essential growth factor responsible for neuroprotection and neurogenesis. Importantly, no difference in the genetic expression of cancer-related genes were identified following MSC-exo treatment indicating for their safety. Conclusions: Our data suggest that adipose-derived MSC-exo carry therapeutic potential in ASD via alternation in gene-expression related mainly to immuno-modulation, reduce neuro-inflammation and increase neuroprotection and neurogenesis. The beneficial effects of the MSC-exo treatment in mice models is being translated to a novel, easy to administer, therapeutic strategy to reduce the symptoms of ASD.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disorder characterized by striatonigral degeneration and olivopontocerebellar atrophy. The main hallmark of MSA is the aggregation of alpha-synuclein in oligodendrocytes, which contributes to the dysfunction and death of the oligodendrocytes, followed by neurodegeneration. Studies suggested that oxidative-excitatory pathway is associated with the progression of the disease. The aim of the current study was to test this concept by overexpression of excitatory amino acid transporter 2, glutamate dehydrogenase and nuclear factor (erythroid-derived 2)-related factor 2 genes in the striatum of two established mouse models of MSA. To induce the first model, we injected the mitochondrial neurotoxin, 3-nitropropionic acid (3-NP), unilaterally into the right striatum in 2-month-old C57BL/6 male mice. We demonstrate a significant improvement in two drug-induced rotational behavior tests, following unilateral injection the three genes. For the second model, we used transgenic mice expressing the alpha-synuclein gene under the proteolipid protein, in the age of 7 months, boosted with 3-NP to enhance the motor deficits and neurodegeneration. We show that the overexpression of the three genes attenuated the motor-related deficit in the elevated bridge and pole tests. Thus, our study indicates that glutamate excito-oxidative toxicity plays a major role in this MSA model and our gene therapy approach might suggest a novel strategy for MSA treatment.

Background CRISPR is a promising novel technology for treating genetic conditions. Therefore, it is essential to further develop and promote treatment’s safety and specificity. While the guide-RNA offers position-specific DNA targeting, it may tolerate small changes such as single-nucleotide polymorphisms (SNPs). To that end, an allele-specific targeting approach is in need for future treatments of heterozygous patients, suffering from genetic conditions caused by a SNP. The SNP-derived PAM approach allows highly allele-specific DNA cleavage by incorporating a protospacer adjacent motif (PAM) sequence only at the target allele. Description Here we present CrisPam, a tool that detects SNP-derived PAMs for allele-specific targeting by the CRISPR/Cas system. The algorithm scans the generation of each reported PAM for a given DNA sequence and its variations. A successful result is such that at least one PAM is generated by a SNP. Thus, the PAM shall be part of the variant allele only and the Cas protein will therefore be able to exclusively bind the variant allele for gene-editing, while the wildtype allele remains unchanged. Conclusion CrisPam is available online for researchers and also offers access to the CrisPamDB, a database that contains the CrisPam analysis for any reported pathogenic SNP in humans.

Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with increased neurodegeneration and vascular impairments. Vascular endothelial growth factor (VEGF), originally described as a key angiogenic factor, has recently been shown to play a crucial role in the nervous system. The objective of this research is to examine the role of VEGF in mediating the apoE4-driven pathologies. We show that hippocampal VEGF levels are lower in apoE4 targeted replacement mice compared to the corresponding apoE3 mice. This effect was accompanied by a specific decrease in both VEGF receptor-2 and HIF1-α. We next set to examine whether upregulation of VEGF can reverse apoE4-driven pathologies, namely the accumulation of hyperphosphorylated tau (AT8) and Aβ42, and reduced levels of the pre-synaptic marker, VGluT1, and of the ApoE receptor, ApoER2. This was first performed utilizing intra-hippocampal injection of VEGF-expressing-lentivirus (LV-VEGF). This revealed that LV-VEGF treatment reversed the apoE4-driven cognitive deficits and synaptic pathologies. The levels of Aβ42 and AT8, however, were increased in apoE3 mice, masking any potential effects of this treatment on the apoE4 mice. Follow-up experiments utilizing VEGF-expressing adeno-associated-virus (AAV-VEGF), which expresses VEGF specifically under the GFAP astrocytic promoter, prevented this effects on apoE3 mice, and reversed the apoE4-related increase in Aβ42 and AT8. Taken together, these results suggest that apoE4-driven pathologies are mediated by a VEGF-dependent pathway, resulting in cognitive impairments and brain pathology. These animal model findings suggest that the VEGF system is a promising target for the treatment of apoE4 carriers in AD.

Background & Aim Background Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. They have been steadily increasing in children over the past several years, with no effective treatment. 2% of all ASD patients, are suffering from a disorder caused by a mutation in shank3 gene. Shank3 is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. During the recent decade, exosomes that derived from mesenchymal stem cells (MSC-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. Objective In order to develop treatment of adipose derived MSC-exo towards clinical trials, we preformed preclinical dose response studies, biodistribution, and intranasal vs intravenous studies. Repeated administration studies were also tested. Here we present results from three different autistic mice models. BTBR model as a multifactorial mice model of autism, and two different Shank3 mutated mice models. The first model is a complete deletion of exon 22 (22q13.3) and the second model is a specific insertion mutation of Guanine to 3680 position in the gene (insG3680) that leads to stop codon. Methods, Results & Conclusion Methods Exosomes were isolated using differential centrifugation protocol and characterized using the 2018MISEV guideline recommendations. Each animal received intranasal administration of 20ul containing 107 exosomes/ul. For intravenous administration, the same number of exosomes, were used, injected in 100ul. Results All three animal models showed significantly improvement in their ASD behavioral phenotypes following intranasal administration. The improvement seems to be dose dependent and was better achieved via intranasal vs intravenous administration. Biodistribution of MSC-exo showed accumulation in the brain within 72 hours, yet reduction of the signal was observed in the kidneys, heart and lungs. Conclusions Our data suggests that adipose derived MSC-exo, carry a therapeutic potential in ASD, via non-invasive intranasal administration in three different mice models. The intranasal administration is especially suitable for ASD pediatric target population. These data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials.

Oxidative stress and inflammation are important players in the pathogenesis of cardiovascular and renal diseases. DJ-1 is a redox-sensitive chaperone that regulates the expression of several antioxidant genes. Activation of the DJ-1/Nrf2 pathway in the kidney inhibits the development and progression of several renal diseases. The 20 aa peptide ND-13 consists of 13 highly conserved aa from the DJ-1 sequence and a TAT- derived 7 aa sequence to help in cell penetration. ND-13 prevents neuronal degeneration in mice; however, its effects on kidney damage remain unknown. We hypothesized that treatment with ND-13 would prevent the renal damage and inflammation associated with unilateral ureter obstruction (UUO). C57Bl/6 mice and DJ1 -/- mice underwent UUO and were divided in 3 groups: control (no UUO), UUO+scrambled peptide (SP) or UUO+ND-13 (3 mg/kg, s.c. daily). After 14 days of treatment, urine and kidneys were collected for analysis of renal damage. ND-13 treatment prevented the development of fibrosis in C57Bl/6 mice (UUO+SP: 702±189% of control, UUO+ND-13: 264±8% of control, n=2-4/group, pTNF- α ( fold change from control: 101±46 in UUO+SP; 18±7 in UUO+ND-13, n=4-5/group, pIL-6 (6.7±2 in UUO+SP; 1.54±0.3 in UUO+ND-13, pTGF- β ( 3.3±1.12 UUO+SP; 1.4±0.03 UUO+ND-13, pColagen1 α 1 (79±16 UUO+SP; 33±3.8 in UUO +ND-13, p-/- mice, treatment with ND-13 similarly decreased expression of TNF-α, IL-6 and TGF-β, but, in contrast, failed to prevent renal fibrosis or kidney expression of col1 α 1 . UUO also led to elevated urinary NGAL, marker of proximal tubular injury, in DJ-1 -/- mice and ND-13 treatment prevented that increase (71±17% UUO vs control: -18±21% UUO+ND-13 vs control, n=5/group). Our results suggest that ND-13 has protective effects on renal injury, fibrosis and inflammation, crucial mechanisms in the pathogenesis of renal disease. Thus, ND-13 treatment may be a new therapeutic approach for the prevention of renal injury, fibrosis and inflammation in renal diseases. Funded by T32DK007545 to CDM and 5P01 HL074940-10, 7R01 DK039308-31 to PAJ.

Autism Spectrum Disorder (ASD) is characterized by persistent deficits in social communication, restricted and repetitive patterns of behavior, interests, or activities and often intellectual disabilities. ASD has a number of prevalent co-morbidities, such as sleep disorders, attention deficit/hyperactivity disorder and epilepsy. No effective treatment for the core symptoms of ASD is currently available. There is increasing interest in cannabinoids, especially cannabidiol (CBD), as monotherapy or add-on treatment for the core symptoms and co-morbidities of ASD. In this review we summarize the available pre-clinical and clinical data regarding the safety and effectiveness of medical cannabis, including CBD, in young ASD patients. Cannabidiol seems to be a candidate for the treatment of ASD. At present, however, there are no convincing pre-clinical or clinical data showing efficacy and safety of cannabinoid treatment in ASD patients.

Cell-to-cell communication system involves Exosomes, small, membrane-enveloped nanovesicles. Exosomes are evolving as effective therapeutic tools for different pathologies. These extracellular vesicles can bypass biological barriers such as the blood-brain barrier, and can function as powerful nanocarriers for drugs, proteins and gene therapeutics. However, to promote exosomes' therapy development, especially for brain pathologies, a better understanding of their mechanism of action, trafficking, pharmacokinetics and bio-distribution is needed. In this research, we established a new method for non-invasive in-vivo neuroimaging of mesenchymal stem cell (MSC)-derived exosomes, based on computed tomography (CT) imaging with glucose-coated gold nanoparticle (GNP) labeling. We demonstrated that the exosomes were efficiently and directly labeled with GNPs, via an energy-dependent mechanism. Additionally, we found the optimal parameters for exosome labeling and neuroimaging, wherein 5 nm GNPs enhanced labeling, and intranasal administration produced superior brain accumulation. We applied our technique in a mouse model of focal ischemia. Imaging and tracking of intranasally-administered GNP-labeled exosomes revealed specific accumulation and prolonged presence at the lesion area, up to 24 hrs. We propose that this novel exosome labeling and in-vivo neuroimaging technique can serve as a general platform for brain theranostics.

The 150-year-long search for treatments of amyotrophic lateral sclerosis (ALS) is still fueled by frustration over the shortcomings of available therapeutics. Contributing to the therapeutic limitations might be the targeting of a single aspect of this multifactorial-multisystemic disease. In an attempt to overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding: EAAT2, GDH2, and NRF2, that act synergistically to address the band and width of the effected excito-oxidative axis, reducing extracellular-glutamate and glutamate availability while improving the metabolic state and the anti-oxidant response. This strategy yielded particularly impressive results, as all three genes together but not separately prolonged survival in ALS mice by an average of 19-22 days. This was accompanied by improvement in every parameter evaluated, including body-weight loss, reflex score, neurologic score, and motor performance. We hope to provide a novel strategy to slow down disease progression and alleviate symptoms of patients suffering from ALS.

Autism spectrum disorders (ASD) are characterized by social communication deficits, cognitive rigidity, and repetitive stereotyped behaviors. Mesenchymal stem cells (MSC) have a paracrine regenerative effect, and were speculated to be a potential therapy for ASD. The BTBR inbred mouse strain is a commonly used model of ASD as it demonstrates robust behavioral deficits consistent with the diagnostic criteria for ASD. BTBR mice also exhibit decreased brain-derived neurotrophic factor (BDNF) signaling and reduced hippocampal neurogenesis. In the current study, we evaluated the behavioral and molecular effects of intracerebroventricular MSC transplantation in BTBR mice. Transplantation of MSC resulted in a reduction of stereotypical behaviors, a decrease in cognitive rigidity and an improvement in social behavior. Tissue analysis revealed elevated BDNF protein levels in the hippocampus accompanied by increased hippocampal neurogenesis in the MSC-transplanted mice compared with sham treated mice. This might indicate a possible mechanism underpinning the behavioral improvement. Our study suggests a novel therapeutic approach which may be translatable to ASD patients in the future.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.

Over the past decade, increasing evidence has implied a significant connection between caspase-6 activity and the pathogenesis of Huntington's disease (HD). Consequently, inhibiting caspase-6 activity was suggested as a promising therapeutic strategy to reduce mutant Huntingtin toxicity, and to provide protection from mutant Huntingtin-induced motor and behavioral deficits. Here, we describe a novel caspase-6 inhibitor peptide based on the huntingtin caspase-6 cleavage site, fused with a cell-penetrating sequence. The peptide reduces mutant Huntingtin proteolysis by caspase-6, and protects cells from mutant Huntingtin toxicity. Continuous subcutaneous administration of the peptide protected pre-symptomatic BACHD mice from motor deficits and behavioral abnormalities. Moreover, administration of the peptide in an advanced disease state resulted in the partial recovery of motor performance, and an alleviation of depression-related behavior and cognitive deficits. Our findings reveal the potential of substrate-based caspase inhibition as a therapeutic strategy, and present a promising agent for the treatment of HD.

Stroke is a leading cause of death worldwide and inflicts serious long-term damage and disability. The vasoconstrictor Endothelin-1, presenting long-term neurological deficits associated with excitotoxicity and oxidative stress is being increasingly used to induce focal ischemic injury as a model of stroke. A DJ-1 based peptide named ND-13 was shown to protect against glutamate toxicity, neurotoxic insults and oxidative stress in various animal models. Here we focus on the benefits of treatment with ND-13 on the functional outcome of focal ischemic injury. Wild type C57BL/6 mice treated with ND-13, after ischemic induction in this model, showed significant improvement in motor function, including improved body balance and motor coordination, and decreased motor asymmetry. We found that DJ-1 knockout mice are more sensitive to Endothelin-1 ischemic insult than wild type mice, contributing thereby additional evidence to the widely reported relevance of DJ-1 in neuroprotection. Furthermore, treatment of DJ-1 knockout mice with ND-13, following Endothelin-1 induced ischemia, resulted in significant improvement in motor functions, suggesting that ND-13 provides compensation for DJ-1 deficits. These preliminary results demonstrate a possible basis for clinical application of the ND-13 peptide to enhance neuroprotection in stroke patients.

BACKGROUND Metal-ion-chelation was suggested to prevent zinc and copper ions-induced amyloid-β (Aβ) aggregation and oxidative stress, both implicated in the pathophysiology of Alzheimer's disease (AD). In a quest for biocompatible metal-ion chelators potentially useful for AD therapy, we previously tested a series of nucleoside 5'-phosphorothioate derivatives as agents for decomposition of Cu(I)/Cu(II)/Zn(II)-Aβ-aggregates, and as inhibitors of OH radicals formation in Cu(I) or Fe(II) /H2O2 solution. Specifically, in our recent study we have identified 2-SMe-ADP(α-S), designated as SAS, as a most promising neuroprotectant. OBJECTIVE To further explore SAS ability to protect the brain from Aβ toxicity both in vitro and in vivo. METHODS We evaluated SAS ability to decompose or inhibit the formation of Aβ42-M(II) aggregates, and rescue primary neurons and astrocytes from Aβ42 toxicity. Furthermore, we aimed at exploring the therapeutic effect of SAS on behavioral and cognitive deficits in the 5XFAD mouse model of AD. RESULTS We found that SAS can rescue primary culture of neurons and astrocytes from Aβ42 toxicity and to inhibit the formation and dissolve Aβ42-Zn(II)/Cu(II) aggregates. Furthermore, we show that SAS treatment can prevent behavioral disinhibition and ameliorate spatial working memory deficits in 5XFAD mice. Notably, the mice were treated at the age of 2 months, before the onset of AD symptoms, for a duration of 2 months, while the effect was demonstrated at the age of 6 months. CONCLUSION Our results indicate that SAS has the potential to delay progression of core pathological characteristics of AD in the 5XFAD mouse model.

Stem cell-based therapies for neurodegenerative diseases aim at halting clinical deterioration by regeneration and by providing local support for damaged tissue. Mesenchymal stem cells (MSCs) hold great potential for cell therapy as they can be efficiently derived from adult tissue, ex vivo expanded in culture and safely transplanted autologously. MSCs were also shown to be able to differentiate toward neural fates and to secrete a broad range of factors able to promote nervous tissue maintenance and repair. Moreover, upon transplantation, MSCs were shown capable of homing toward lesioned areas, implying their potential use as vehicles for therapeutic agents administration. Indeed, various advantageous effects were reported following human MSCs transplantation into rodent models of neurodegenerative diseases, such as neurotrophic factor-mediated protection, enhanced neurogenesis, modulation of inflammation, and abnormal protein aggregate clearance. Per journal style, most nonstandard abbreviations must be used at least two times in the abstract to be retained; NTF was used once and thus has been deleted. Recent studies have also used ex vivo manipulation for enhanced expression of potentially favorable factors, by so exploiting the homing capacity of MSCs for effective expression at the lesion site. Here, we will summarize current advancements in MSCs-based therapies for neurodegenerative diseases. We will examine the roles of central mechanisms suggested to mediate the beneficial effects of MSCs-based therapy and consider the augmentation of these mechanisms for superior clinical outcomes in rodent models of neurodegeneration as well as in clinical trials. Stem Cells 2017;35:1867-1880.

The discovery of neurogenesis in the adult brain has created new possibilities for therapeutics in neurodegenerative diseases. Neural precursor cells, which have been found in various parts of the brain, e.g., the subventricular zone (SVZ) and substantia nigra (SN), have promising potential to replace the extensive loss of neurons occurring in neurodegenerative disorders. In Parkinson’s disease (PD) the degeneration of nigral dopaminergic neurons and consequently the nigrostriatal pathway, which has been found to innervate proximally to the SVZ, causes motor and cognitive impairments. There is strong evidence that neurogenesis is impaired in PD, which has been related to the nonmotor symptoms of the disease. Recent evidence supports that this impairment in neurogenesis is partially caused by the lack of dopamine in one of the adult neurogenic niches, the SVZ. Thus, restoring the dopaminergic pathway in PD patients may have implications not only for motor function improvement, but for other cognitive and autonomic symptoms. Currently, there are no effective treatments that can stop or reverse the neurodegeneration process in the brain. Here we review the neurogenic process and observed alterations found in PD animal models and postmortem brains of PD patients. Finally, we review several attempts to stimulate the neurogenic process for nigral and/or striatal dopaminergic restoration by transgenic expression, exercise, or cell therapy. J. Comp. Neurol. 000:000–000, 2014.

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

Alzheimer's disease (AD) affects 13% of the population over the age of 65. Behavioral and neuropsychiatric symptoms are frequent and affect 80% of patients. Adult hippocampal neurogenesis, which is impaired in AD, is involved in learning and memory. It remains unclear, however, whether increasing adult neurogenesis improves behavioral symptoms in AD. We report that in the 3xTgAD mouse model of AD, chronic Wnt3a overexpression in the ventral hippocampus dentate gyrus (DG) restored adult neurogenesis to physiological levels. The restoration of adult neurogenesis led to full recovery of danger assessment impairment and the effect was blocked by ablation of neurogenesis with X-irradiation. Finally, using a bed nucleus of stria terminalis (BNST) mRNA expression array, we found that the expression of the 5-HT1A receptor in 3xTgAD mice is selectively decreased and normalized by Wnt3a overexpression in the ventral hippocampus DG, and this normalization is neurogenesis dependent. These findings indicate that reestablishing a functional population of hippocampal newborn neurons in adult AD mice rescues behavioral symptoms, suggesting that adult neurogenesis may be a promising therapeutic target for alleviating behavioral deficits in AD patients.

Human oral mucosa stem cells (hOMSC) are a recently described neural crest-derived stem cell population. Therapeutic quantities of potent hOMSC can be generated from small biopsies obtained by minimally invasive procedures. Our objective was to evaluate the potential of hOMSC to differentiate into astrocyte-like cells and provide peripheral neuroprotection. We induced hOMSC differentiation into cells showing an astrocyte-like morphology that expressed characteristic astrocyte markers as glial fibrillary acidic protein, S100β, and the excitatory amino acid transporter 1 and secreted neurotrophic factors (NTF) such as brain-derived neurotrophic factor, vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1. Conditioned medium of the induced cells rescued motor neurons from hypoxia or oxidative stress in vitro, suggesting a neuroprotective effect mediated by soluble factors. Given the neuronal support (NS) ability of the cells, the differentiated cells were termed hOMSC-NS. Rats subjected to sciatic nerve injury and transplanted with hOMSC-NS showed improved motor function after transplantation. At the graft site we found the transplanted cells, increased levels of NTF, and a significant preservation of functional neuromuscular junctions, as evidenced by colocalization of α-bungarotoxin and synaptophysin. Our findings show for the first time that hOMSC-NS generated from oral mucosa exhibit neuroprotective effects in vitro and in vivo and point to their future therapeutic use in neural disorders.

OBJECTIVES. Disrupted in schizophrenia 1 (DISC1) is considered the most prominent candidate gene for schizophrenia. In this study, we aimed to characterize behavioural and brain biochemical traits in a mouse expressing a dominant negative DISC1mutant (DN-DISC1).DN-DISC1 mice underwent behavioural tests to evaluate object recognition, social preference and social novelty seeking. ELISA was conducted on brain tissue to evaluate BDNF levels. Western blot was employed to measure BDNF receptor (TrkB) and cannabinoid receptor CB1.The mutant DISC1 mice displayed deficits in preference to social novelty while both social preference and object recognition were intact. Biochemical analysis of prefrontal cortex and hippocampus revealed a modest reduction in cortical TrkB protein levels of male mice while no differences in BDNF levels were observed. We found sex dependent differences in the expression of cannabinoid-1 receptors.We describe novel behavioural and biochemical abnormalities in the DN-DISC1 mouse model of schizophrenia. The data shows for the first time a possible link between DISC1 mutation and the cannabinoid system.

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine strategies in brain diseases. Experimental studies have shown that repeated administration of phencyclidine (PCP) leads to schizophrenia-like behavioral changes in mice. The aim of the present study was to explore the effectiveness of MSC transplantation into the hippocampus in attenuating PCP-induced social behavior deficits. PCP was administered subcutaneously to C57bl mice (10 mg/kg daily) for 2 weeks. On the first day of PCP administration, adult human MSCs were transplanted into the hippocampus. A week after the last PCP dose, the mice underwent social preference testing. MSC transplantation was associated with a significant reduction in the adverse social behavior induced by PCP. Immunohistochemical analysis revealed that the stem cells survived in the mouse brain, and hippocampal Western blot analysis revealed a statistical trend towards a decrease in cleaved caspase 3 protein levels in the stem cell treated group. Upon in vitro co-culture of astrocytes and MSCs, the MSCs, in the presence of PCP, positively regulated astrocyte expression of genes involved in glutamate metabolism and antioxidant defenses. These findings suggest that MSC transplantation into the hippocampus may serve as a novel neuroprotective tool for the treatment of the PCP-induced schizophrenia-like social endophenotype. The mechanism underlying the beneficial behavioral effect may involve modulation of host astrocyte functioning, including glutamate processing and antioxidant capacity.