Neural Cells for Neurodegenerative Diseases in Clinical Trials.

Neurodegenerative diseases (ND) are an entire spectrum of clinical conditions that affect the central and peripheral nervous system. There is no cure currently, with treatment focusing mainly on slowing down progression or symptomatic relief. Cellular therapies with various cell types from different sources are being conducted as clinical trials for several ND diseases. They include neural, mesenchymal and hemopoietic stem cells, and neural cells derived from embryonic stem cells and induced pluripotent stem cells. In this review, we present the list of cellular therapies for ND comprising 33 trials that used neural stem progenitors, 8 that used differentiated neural cells ,and 109 trials that involved non-neural cells in the 7 ND. Encouraging results have been shown in a few early-phase clinical trials that require further investigations in a randomized setting. However, such definitive trials may not be possible given the relative cost of the trials, and in the setting of rare diseases.

Emerging Intrinsic Therapeutic Targets for Metastatic Breast Cancer.

Breast cancer is now the most common cancer worldwide, and it is also the main cause of cancer-related death in women. Survival rates for female breast cancer have significantly improved due to early diagnosis and better treatment. Nevertheless, for patients with advanced or metastatic breast cancer, the survival rate is still low, reflecting a need for the development of new therapies. Mechanistic insights into metastatic breast cancer have provided excellent opportunities for developing novel therapeutic strategies. Although high-throughput approaches have identified several therapeutic targets in metastatic disease, some subtypes such as triple-negative breast cancer do not yet have an apparent tumor-specific receptor or pathway to target. Therefore, exploring new druggable targets in metastatic disease is a high clinical priority. In this review, we summarize the emerging intrinsic therapeutic targets for metastatic breast cancer, including cyclin D-dependent kinases CDK4 and CDK6, the PI3K/AKT/mTOR pathway, the insulin/IGF1R pathway, the EGFR/HER family, the JAK/STAT pathway, poly(ADP-ribose) polymerases (PARP), TROP-2, Src kinases, histone modification enzymes, activated growth factor receptors, androgen receptors, breast cancer stem cells, matrix metalloproteinases, and immune checkpoint proteins. We also review the latest development in breast cancer immunotherapy. Drugs that target these molecules/pathways are either already FDA-approved or currently being tested in clinical trials.

The Combined Effects of Topography and Stiffness on Neuronal Differentiation and Maturation Using a Hydrogel Platform.

Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2-10 µm) were patterned onto gels with varying stiffness (6.1-110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of ß-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.

Maternal Methamphetamine Exposure Influences Behavioral Sensitization and Nucleus Accumbens DNA Methylation in Subsequent Generation.

The deleterious effects of methamphetamine (METH) exposure extend beyond abusers, and may potentially impact the vulnerability of their offspring in developing addictive behaviors. Epigenetic signatures have been implicated in addiction, yet the characteristics to identify prenatal METH abuse to offspring addiction risk remains elusive. Here, we used escalating doses of METH-exposed mouse model in F0 female mice before and during pregnancy to simulate the human pattern of drug abuse and generated METH-induced behavioral sensitization to investigate the addictive behavior in offspring mice. We then utilized whole genome-bisulfite sequencing (WGBS) to investigate the methylation signature of nucleus accumbens (NAc) in male METH-sensitized mice. Interestingly, male but not female offspring exhibited an enhanced response to METH-induced behavioral sensitization. Additionally, the METH-exposed group of male mice underwent a more comprehensive wave of epigenome remodeling over all genomic elements compared with unexposed groups due to drug exposure history. 104,219 DMCs (METH-SAL vs. SAL-SAL) induced by prenatal METH-exposure were positively correlated with that of postnatal METH-exposure (38,570, SAL-METH vs. SAL-SAL). Moreover, 4,983 DMCs induced by pre- and postnatal METH exposure (METH-METH vs. SAL-METH) were negatively correlated with that of postnatal METH exposure, and 371 commonly changed DMCs between the two comparison groups also showed a significantly negative correlation and 86 annotated genes functionally enriched in the pathways of neurodevelopment and addiction. Key annotated genes included Kirrel3, Lrpprc, and Peg3, implicated in neurodevelopmental processes, were down-regulated in METH-METH group mice compared with the SAL-METH group. Taken together, we render novel insights into the epigenetic correlation of drug exposure and provide evidence for epigenetic characteristics that link maternal METH exposure to the intensity of the same drug-induced behavioral sensitization in adult offspring.

Alzheimer's Disease Blood Biomarkers Associated With Neuroinflammation as Therapeutic Targets for Early Personalized Intervention.

The definitive diagnosis of Alzheimer's Disease (AD) without the need for neuropathological confirmation remains a challenge in AD research today, despite efforts to uncover the molecular and biological underpinnings of the disease process. Furthermore, the potential for therapeutic intervention is limited upon the onset of symptoms, providing motivation for studying and treating the AD precursor mild cognitive impairment (MCI), the prodromal stage of AD instead. Applying machine learning classification to transcriptomic data of MCI, AD, and cognitively normal (CN) control patients, we identified differentially expressed genes that serve as biomarkers for the characterization and classification of subjects into MCI or AD groups. Predictive models employing these biomarker genes exhibited good classification performances for CN, MCI, and AD, significantly above random chance. The PI3K-Akt, IL-17, JAK-STAT, TNF, and Ras signaling pathways were also enriched in these biomarker genes, indicating their diagnostic potential and pathophysiological roles in MCI and AD. These findings could aid in the recognition of MCI and AD risk in clinical settings, allow for the tracking of disease progression over time in individuals as part of a therapeutic approach, and provide possible personalized drug targets for early intervention of MCI and AD.

A novel microRNA, novel-m009C, regulates methamphetamine rewarding effects.

Methamphetamine (METH) is a widely abused psychostimulant, whose hyper-rewarding property is believed to underlie its addictive effect, but the molecular mechanism regulating this effect remains unclear. We previously reported that decreased expression of a novel microRNA (miRNA), novel-m009C, is implicated in the regulation of METH hyperlocomotion. Here, we found that novel-m009C may be homologous to hsa-miR-604. Its expression is consistently downregulated in the nucleus accumbens (NAc) of mice when exposed to METH and cocaine, whereas significant alterations in novel-m009C expression were not observed in the NAc of mice subjected to other rewarding and psychiatric stimuli, such as sucrose, morphine and MK-801. We further found the substantial reduction in novel-m009C expression may be regulated by both dopamine receptor D1 (D1R) and D2 (D2R). Increasing novel-m009C levels in the NAc attenuated METH-induced conditioned place preference (CPP) and hyperlocomotion, whereas inhibiting novel-m009C expression in the NAc enhanced these effects but did not change the preference of mice for a natural reward, i.e., sucrose. These effects may involve targeting of genes important for the synaptic transmission, such as Grin1 (NMDAR subunit 1). Our findings demonstrate an important role for NAc novel-m009C in regulating METH reward, reveal a novel molecular regulator of the actions of METH on brain reward circuitries and provide a new strategy for treating METH addiction based on the modulation of small non-coding RNAs.

WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons.

The activation of chloride (Cl-)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental "switch" in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl- concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13-15) but had no effect on immature neurons (DIV6-8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA . Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the "adult" GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

Potential Ago2/miR-3068-5p Cascades in the Nucleus Accumbens Contribute to Methamphetamine-Induced Locomotor Sensitization of Mice.

Dysregulation of microRNA (miRNA) biogenesis is involved in drug addiction. Argonaute2 (Ago2), a specific splicing protein involved in the generation of miRNA, was found to be dysregulated in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice in our previous study. Here, we determined whether Ago2 in the NAc regulates METH sensitization in mice and identified Ago2-dependent miRNAs involved in this process. We found a gradual reduction in Ago2 expression in the NAc following repeated METH use. METH-induced hyperlocomotor activity in mice was strengthened by knocking down NAc neuronal levels of Ago2 but reduced by overexpressing Ago2 in NAc neurons. Surprisingly, miR-3068-5p was upregulated following overexpression of Ago2 and downregulated by silencing Ago2 in the NAc. Knocking down miR-3068-5p, serving as an Ago2-dependent miRNA, strengthened the METH sensitization responses in mice. These findings demonstrated that dysregulated Ago2 in neurons in the NAc is capable of regulating METH sensitization and suggested a potential role of Ago2-dependent miR-3068-5p in METH sensitization.

Regulation of miR-128 in the nucleus accumbens affects methamphetamine-induced behavioral sensitization by modulating proteins involved in neuroplasticity.

Methamphetamine (METH) -induced behavioral sensitization depends on long-term neuroplasticity in the mesolimbic dopamine system, especially in the nucleus accumbens (NAc). miR-128, a brain enriched miRNA, was found to have abilities in regulating neuronal excitability and formation of fear-extinction memory. Here, we aim to identify the role of miR-128 on METH-induced locomotor sensitization of male mice. We identified a significant increase of miR-128 in the NAc of mice upon repeated-intermittent METH exposure but not acute METH administration. Microinjection of adeno-associated virus (AAV)-miR-128 over-expression and inhibition constructs into the NAc of mice resulted in enhanced METH-induced locomotor sensitization and attenuated effects of METH respectively. Isobaric tags for relative and absolute quantification (iTRAQ) technology and ingenuity pathway analysis (IPA) were carried out to uncover the potential molecular mechanisms underlying miR-128-regulated METH sensitization. Differentially expressed proteins, including 25 potential targets for miR-128 were annotated in regulatory pathways that modulate dendritic spines, synaptic transmission and neuritogenesis. Of which, Arf6, Cpeb3 and Nlgn1, were found to be participating in miR-128-regulated METH sensitization. Consistently, METH-induced abnormal changes of Arf6, Cpeb3 and Nlgn1 in the NAc of mice were also detected by qPCR and validated by western blot analysis. Thus, miR-128 may contribute to METH sensitization through controlling neuroplasticity. Our study suggested miR-128 was an important regulator of METH- induced sensitization and also provided the potential molecular networks of miR-128 in regulating METH-induced sensitization.

MeCP2 Dysfunction in Rett Syndrome and Neuropsychiatric Disorders.

Elucidating the functions of a particular gene is paramount to the understanding of how its dysfunction contributes to disease. This is especially important when the gene is implicated in multiple different disorders. One such gene is methyl-CpG-binding protein 2 (MECP2), which has been most prominently associated with the neurodevelopmental disorder Rett syndrome, as well as major neuropsychiatric disorders such as autism and schizophrenia. Being initially identified as a transcriptional regulator that modulates gene expression and subsequently also shown to be involved in other molecular events, dysfunction of the MeCP2 protein has the potential to affect many cellular processes. In this chapter, we will briefly review the functions of the MeCP2 protein and how its mutations are implicated in Rett syndrome and other neuropsychiatric disorders. We will further discuss about the mouse models that have been generated to specifically dissect the function of MeCP2 in different cell types and brain regions. It is envisioned that such thorough and targeted examination of MeCP2 functions can aid in enlightening the role that it plays in normal and dysfunctional physiological systems.

Behavioral Characterization of MeCP2 Dysfunction-Associated Rett Syndrome and Neuropsychiatric Disorders.

The methyl-CpG-binding protein 2 (MECP2) gene has been implicated in multiple neuropsychiatric disorders such as autism and schizophrenia and, most notably, Rett syndrome (RTT). Mouse models of MeCP2 dysfunction that have been developed are thus important not only for examining the protein's contribution to RTT, but also for elucidating the etiologies of other MECP2-associated neuropsychiatric disorders. In this chapter, we present protocols for three behavioral assays for characterizing major functional domains of MeCP2 dysfunction-the open field test for measuring general locomotor activity and anxiety-like behavior, the three-chambered Crawley box test for assessing social preference and social novelty, and the rotarod assay for testing locomotor coordination. It is hoped that these information facilitate systematic characterization of mouse models that may aid in elucidating the role of MeCP2 in neurological disorders, as well as assessing the effects of putative mechanistic and therapeutic interventions.

Correction to: Rescue of Methyl-CpG Binding Protein 2 Dysfunction-induced Defects in Newborn Neurons by Pentobarbital.

C.-H. Yang's appeared incorrectly on the original publication of this article.

Cell surface α2,3-linked sialic acid facilitates Zika virus internalization.

The emergence of neurotropic Zika virus (ZIKV) raised a public health emergency of global concern. ZIKV can cross the placental barrier and infect foetal brains, resulting in microcephaly, but the pathogenesis of ZIKV is poorly understood. With recent findings reporting AXL as a type I interferon antagonist rather than an entry receptor, the exact entry mechanism remains unresolved. Here we report that cell surface sialic acid plays an important role in ZIKV infection. Removal of cell surface sialic acid by neuraminidase significantly abolished ZIKV infection in Vero cells and human induced-pluripotent stem cells-derived neural progenitor cells. Furthermore, knockout of the sialic acid biosynthesis gene encoding UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase resulted in significantly less ZIKV infection of both African and Asian lineages. Huh7 cells deficient in α2,3-linked sialic acid through knockout of ST3 ß-galactoside-α2,3-sialyltransferase 4 had significantly reduced ZIKV infection. Removal of membrane-bound, un-internalized virus with pronase treatment revealed the role of sialic acid in ZIKV internalization but not attachment. Sialyllactose inhibition studies showed that there is no direct interaction between sialic acid and ZIKV, implying that sialic acid could be mediating ZIKV-receptor complex internalization. Identification of α2,3-linked sialic acid as an important host factor for ZIKV internalization provides new insight into ZIKV infection and pathogenesis.

Ago2 and Dicer1 are involved in METH-induced locomotor sensitization in mice via biogenesis of miRNA.

microRNA (miRNA) play important roles in drug addiction and act as a post-transcriptional regulator of gene expression. We previously reported extensive downregulation of miRNAs in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice. However, the regulatory mechanism of this METH-induced downregulation of miRNAs has yet to be elucidated. Thus, we examined METH-induced changes in the expression of miRNAs and their precursors, as well as the expression levels of mRNA and the proteins involved in miRNA biogenesis such as Dicer1 and Ago2, in the nucleus accumbens of METH-induced locomotor sensitized mice. miRNAs and Ago2 were significantly downregulated, while the expression of miRNA precursors remained unchanged or upregulated, which suggests that the downregulation of miRNAs was likely due to a reduction in Ago2-mediated splicing but unlikely to be regulated at the transcription level. Interestingly, the expression level of Dicer1, which is a potential target of METH-induced decreased miRNAs, such as miR-124, miR-212 and miR-29b, was significantly increased. In conclusion, this study indicates that miRNA biogenesis (such as Ago2 and Dicer1) and their miRNA products may have a role in the development of METH addiction.

Choline Rescues Behavioural Deficits in a Mouse Model of Rett Syndrome by Modulating Neuronal Plasticity.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls, with 95% of RTT cases resulting from mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Choline, a dietary micronutrient found in most foods, has been shown to be important for brain development and function. However, the exact effects and mechanisms are still unknown. We found that 13 mg/day (1.7 × required daily intake) of postnatal choline treatment to Mecp2-conditional knockout mice rescued not only deficits in motor coordination, but also their anxiety-like behaviour and reduced social preference. Cortical neurons in the brains of Mecp2-conditional knockout mice supplemented with choline showed enhanced neuronal morphology and increased density of dendritic spines. Modelling RTT in vitro by knocking down the expression of the MeCP2 protein with shRNA, we found that choline supplementation to MeCP2-knockdown neurons increased their soma sizes and the complexity of their dendritic arbors. Rescue of the morphological defects could lead to enhanced neurotransmission, as suggested by an observed trend of increased expression of synaptic proteins and restored miniature excitatory postsynaptic current frequency in choline-supplemented MeCP2-knockdown neurons. Through the use of specific inhibitors targeting each of the known physiological pathways of choline, synthesis of phosphatidylcholine from choline was found to be essential in bringing about the changes seen in the choline-supplemented MeCP2-knockdown neurons. Taken together, these data reveal a role of choline in modulating neuronal plasticity, possibly leading to behavioural changes, and hence, a potential for using choline to treat RTT.

Maternal methamphetamine exposure causes cognitive impairment and alteration of neurodevelopment-related genes in adult offspring mice.

Prenatal drug exposure altered cognitive function in individuals, and may also impact their offspring's susceptibility to cognitive impairment. The high incidence of methamphetamine (METH) abuse among adolescents and women of childbearing age elevates the importance to determine the influence of maternal METH exposure on cognitive functions in the descendants. We hypothesized that maternal METH exposure affects cognitive behavior in offspring mice by disrupting gene expression associated with neural development. Here, female C57BL/6 mice were exposed to intermittent escalating doses of METH or saline from adolescence to adulthood, and then continued through pregnancy. Interestingly, male but not female offspring exhibited impaired short-term recognition memory and long-term spatial memory retention in novel object recognition and Morris water maze test respectively. Additionally, maternal METH exposure altered neurodevelopmental genes in both male and female offspring, and 12 differentially expressed genes between male and female were observed in the HPC and NAc regions. These differentially expressed genes are involved in neurogenesis, axon guidance, neuron migration and synapse of neural development circuits. Our observations suggest that maternal METH exposure induced differential expression patterns of neurodevelopment-related genes in the HPC and NAc of male and female mice, which may underlie the different cognitive behavior phenotypes in both genders.

Sequential Application of Discrete Topographical Patterns Enhances Derivation of Functional Mesencephalic Dopaminergic Neurons from Human Induced Pluripotent Stem Cells.

Parkinson's Disease is a progressive neurodegenerative disorder attributed to death of mesencephalic dopaminergic (DA) neurons. Pluripotent stem cells have great potential in the study for this late-onset disease, but acquirement of cells that are robust in quantity and quality is still technically demanding. Biophysical cues have been shown to direct stem cell fate, but the effect of different topographies in the lineage commitment and subsequent maturation stages of cells have been less examined. Using human induced pluripotent stem cells (iPSCs), we applied topographical patterns sequentially during differentiation stages and examined their ability to influence derivation yield and functionality of regionalized subtype-specific DA neurons. Gratings showed higher yield of DA neurons and may be beneficial for initial lineage commitment. Cells derived on pillars in the terminal differentiation stage have increased neuronal complexity, and were more capable of firing repetitive action potentials, showing that pillars yielded better network formation and functionality. Our topography platform can be applied to patient-derived iPSCs as well, and that cells harbouring LRRK2 mutation were more functionally mature when optimal topographies were applied sequentially. This will hopefully accelerate development of robust cell models that will provide novel insights into discovering new therapeutic approaches for Parkinson's Disease.

Rab23 and developmental disorders.

Rab23 is a conserved member of the Rab family of small GTPases that regulates membrane trafficking in eukaryotes. It is unique amongst the Rabs in terms of its implicated role in mammalian development, as originally illustrated by the embryonic lethality and open neural tube phenotype of a spontaneous mouse mutant that carries homozygous mutation of open brain, a gene encoding Rab23. Rab23 was initially identified to act as an antagonist of Sonic hedgehog (Shh) signaling, and has since been implicated in a number of physiological and pathological roles, including oncogenesis. Interestingly, RAB23 null allele homozygosity in humans is not lethal, but instead causes the developmental disorder Carpenter's syndrome (CS), which is characterized by craniofacial malformations, polysyndactyly, obesity and intellectual disability. CS bears some phenotypic resemblance to a spectrum of hereditary defects associated with the primary cilium, or the ciliopathies. Recent findings have in fact implicated Rab23 in protein traffic to the primary cilium, thus linking it with the primary cellular locale of Shh signaling. Rab23 also has Shh and cilia-independent functions. It is known to mediate the expression of Nodal at the mouse left lateral plate mesoderm and Kupffer's vesicle, the zebrafish equivalent of the mouse node. It is thus important for the left-right patterning of vertebrate embryos. In this review, we discuss the developmental disorders associated with Rab23 and attempt to relate its cellular activities to its roles in development.

Rab23 Regulates Radial Migration of Projection Neurons via N-cadherin.

Radial migration of cortical projection neurons is a prerequisite for shaping a distinct multilayered cerebral cortex during mammalian corticogenesis. Members of Rab GTPases family were reported to regulate radial migration. Here, in vivo conditional knockout or in utero knockdown (KD) of Rab23 in mice neocortex causes aberrant polarity and halted migration of cortical projection neurons. Further investigation of the underlying mechanism reveals down-regulation of N-cadherin in the Rab23-deficient neurons, which is a cell adhesion protein previously known to modulate radial migration. (Shikanai M, Nakajima K, Kawauchi T. 2011. N-cadherin regulates radial glial fiber-dependent migration of cortical locomoting neurons. Commun Integr Biol. 4:326-330.) Interestingly, pharmacological inhibition of extracellular signal-regulated kinases (ERK1/2) also decreases the expression of N-cadherin, implicating an upstream effect of ERK1/2 on N-cadherin and also suggesting a link between Rab23 and ERK1/2. Further biochemical studies show that silencing of Rab23 impedes activation of ERK1/2 via perturbed platelet-derived growth factor-alpha (PDGFRα) signaling. Restoration of the expression of Rab23 or N-cadherin in Rab23-KD neurons could reverse neuron migration defects, indicating that Rab23 modulates migration through N-cadherin. These studies suggest that cortical neuron migration is mediated by a molecular hierarchy downstream of Rab23 via N-cadherin.