CXCR2 increases in ALS cortical neurons and its inhibition prevents motor neuron degeneration in vitro and improves neuromuscular function in SOD1G93A mice.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by depletion of motor neurons (MNs), for which effective medical treatments are still required. Previous transcriptomic analysis revealed the up-regulation of C-X-C motif chemokine receptor 2 (CXCR2)-mRNA in a subset of sporadic ALS patients and SOD1G93A mice. Here, we confirmed the increase of CXCR2 in human ALS cortex, and showed that CXCR2 is mainly localized in cell bodies and axons of cortical neurons. We also investigated the effects of reparixin, an allosteric inhibitor of CXCR2, in degenerating human iPSC-derived MNs and SOD1G93A mice. In vitro, reparixin rescued MNs from apoptotic cell death, preserving neuronal morphology, mitochondrial membrane potential and cytoplasmic membrane integrity, whereas in vivo it improved neuromuscular function of SOD1G93A mice. Altogether, these data suggest a role for CXCR2 in ALS pathology and support its pharmacological inhibition as a candidate therapeutic strategy against ALS at least in a specific subgroup of patients.

iPSCs: A Preclinical Drug Research Tool for Neurological Disorders.

The development and commercialization of new drugs is an articulated, lengthy, and very expensive process that proceeds through several steps, starting from target identification, screening new leading compounds for testing in preclinical studies, and subsequently in clinical trials to reach the final approval for therapeutic use. Preclinical studies are usually performed using both cell cultures and animal models, although they do not completely resume the complexity of human diseases, in particular neurodegenerative conditions. To this regard, stem cells represent a powerful tool in all steps of drug discovery. The recent advancement in induced Pluripotent Stem Cells (iPSCs) technology has opened the possibility to obtain patient-specific disease models for drug screening and development. Here, we report the use of iPSCs as a disease model for drug development in the contest of neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD), Amyotrophic lateral Sclerosis (ALS), and Fragile X syndrome (FRAX).

Dental mesenchymal stem cells and neuro-regeneration: a focus on spinal cord injury.

Regenerative medicine is a branch of translational research that aims to reestablish irreparably damaged tissues and organs by stimulating the body's own repair mechanisms via the implantation of stem cells differentiated into specialized cell types. A rich source of adult stem cells is located inside the tooth and is represented by human dental pulp stem cells, or hDPSCs. These cells are characterized by a high proliferative rate, have self-renewal and multi-lineage differentiation properties and are often used for tissue engineering and regenerative medicine. The present review will provide an overview of hDPSCs and related features with a special focus on their potential applications in regenerative medicine of the nervous system, such as, for example, after spinal cord injury. Recent advances in the identification and characterization of dental stem cells and in dental tissue engineering strategies suggest that bioengineering approaches may successfully be used to regenerate districts of the central nervous system, previously considered irreparable.

PACAP and PAC1R are differentially expressed in motor cortex of amyotrophic lateral sclerosis patients and support survival of iPSC-derived motor neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal and disabling neurodegenerative disease characterized by upper and lower motor neurons depletion. In our previous work, comprehensive genomic profiling of 41 motor cortex samples enabled to discriminate controls from sporadic ALS patients, and segregated these latter into two distinct subgroups (SALS1 and SALS2), each associated with different deregulated genes. In the present study, we focused our attention on two of them, Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its type 1 receptor (PAC1R), and validated the results of the transcriptome experiments by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunohistochemistry and Western blot analysis. To assess the functional role of PACAP and PAC1R in ALS, we developed an in vitro model of human induced pluripotent stem cells (iPSC)-derived motor neurons and examined the trophic effects of exogenous PACAP following neurodegenerative stimuli. Treatment with 100 nm PACAP was able to effectively rescue iPSC-derived motor neurons from apoptosis, as shown by cell viability assay and protein dosage of the apoptotic marker (BAX). All together, these data suggest that perturbations in the PACAP-PAC1R pathway may be involved in ALS pathology and represent a potential drug target to enhance motor neuron viability.

NeuroArray, A Custom CGH Microarray to Decipher Copy Number Variants in Alzheimer's Disease.

BACKGROUND: Copy Number Variants (CNVs) represent a prevailing type of structural variation (deletions or duplications) in the human genome. In the last few years, several studies have demonstrated that CNVs represent significant mutations in Alzheimer's Disease (AD) hereditability. Currently, innovative high-throughput platforms and bioinformatics algorithms are spreading to screening CNVs involved in different neurological diseases. In particular, the use of custom arrays, based on libraries of probes that can detect significant genomic regions, have greatly improved the resolution of targeted regions and the identification of chromosomal aberrations. OBJECTIVE: In this work, we report the use of NeuroArray, a custom CGH microarray useful to screening and further investigate the role of the recurring genomic aberrations in patients with confirmed or suspected AD. METHODS: The custom oligonucleotide aCGH design includes 641 genes and 9118 exons, linked to AD. The genomic DNA was isolated from blood samples of AD affected patients. The entire protocol of custom NeuroArray included digestion, labelling and hybridization steps as a standard aCGH assay. RESULTS: The NeuroArray analysis revealed the presence of amplifications in several genes associated with AD. In the coding regions of these genes, 14,586 probes were designed with a 348 bp median probe spacing. The majority of targeted AD genes map on chromosomes 1 and 10. A significant aspect of the NeuroArray design is that 95% of the total exon targets is covered by at least one probe, a resolution higher than CGH array platforms commercially available. CONCLUSION: By identifying with a high sensitivity the chromosomal abnormalities in a large panel of AD-related genes and other neurological diseases, the NeuroArray platform is a valid tool for clinical diagnosis.

Genotyping of KRAS Mutational Status by the In-Check Lab-on-Chip Platform.

The KRAS oncogene is involved in the pathogenesis of several types of cancer, particularly colorectal cancer (CRC). The most frequent mutations in this gene are associated with poor survival, increased tumor aggressiveness and resistance to therapy with anti-epidermal growth factor receptor (EGFR) antibodies. For this reason, KRAS mutation testing has become increasingly common in clinical practice for personalized cancer treatments of CRC patients. Detection methods for KRAS mutations are currently expensive, laborious, time-consuming and often lack of diagnostic sensitivity and specificity. In this study, we describe the development of a Lab-on-Chip assay for genotyping of KRAS mutational status. This assay, based on the In-Check platform, integrates microfluidic handling, a multiplex polymerase chain reaction (PCR) and a low-density microarray. This integrated sample-to-result system enables the detection of KRAS point mutations, including those occurring in codons 12 and 13 of exon 2, 59 and 61 of exon 3, 117 and 146 of exon 4. Thanks to its miniaturization, automation, rapid analysis, minimal risk of sample contamination, increased accuracy and reproducibility of results, this Lab-on-Chip platform may offer immediate opportunities to simplify KRAS genotyping into clinical routine.

Miniaturized Real-Time PCR on a Q3 System for Rapid KRAS Genotyping.

Colorectal cancer (CRC) is an aggressive human malignancy with a complex genomic landscape harboring KRAS mutations. In 40%-60% of patients with CRC, constantly active KRAS proteins affect the prognosis, surgical strategy, and clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) agents. For this reason, there is a greater demand for minimally-invasive diagnostic devices to characterize the genetic pattern and prevent the acquired mechanism to drug resistance. The rapid developments in cutting-edge diagnostic techniques are expected to play a growing role in medicine and represent an attractive promise to identify potential responders to personalized medicine. Here we propose a new method to simultaneously detect the main KRAS mutations on the portable real-time PCR Q3 platform. This platform is based on hybrid silicon-plastic technology implemented in a miniaturized chip able to achieve a sample-in answer-out rapid analysis, allowing a new approach to genetic counseling and testing.

Increasing the Coding Potential of Genomes Through Alternative Splicing: The Case of PARK2 Gene.

The completion of the Human Genome Project aroused renewed interest in alternative splicing, an efficient and widespread mechanism that generates multiple protein isoforms from individual genes. Although our knowledge about alternative splicing is growing exponentially, its real impact on cellular life is still to be clarified. Connecting all splicing features (genes, splice transcripts, isoforms, and relative functions) may be useful to resolve this tangle. Herein, we will start from the case of a single gene, Parkinson protein 2, E3 ubiquitin protein ligase (PARK2), one of the largest in our genome. This gene is implicated in the pathogenesis of autosomal recessive juvenile Parkinsonism and it has been recently linked to cancer, leprosy, autism, type 2 diabetes mellitus and Alzheimer's disease. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification and protein diversity in tissues and cells. This review will provide an update of all human PARK2 alternative splice transcripts and isoforms presently known, and correlate them to those in rat and mouse, two common animal models for studying human disease genes. Alternative splicing relies upon a complex process that could be easily altered by both cis and trans-acting mutations. Although the contribution of PARK2 splicing in human disease remains to be fully explored, some evidences show disruption of this versatile form of genetic regulation may have pathological consequences.

Stem Cells: Innovative Therapeutic Options for Neurodegenerative Diseases?

Neurodegenerative diseases are characterized by the progressive loss of structure and/or function of both neurons and glial cells, leading to different degrees of pathology and loss of cognition. The hypothesis of circuit reconstruction in the damaged brain via direct cell replacement has been pursued extensively so far. In this context, stem cells represent a useful option since they provide tissue restoration through the substitution of damaged neuronal cells with exogenous stem cells and create a neuro-protective environment through the release of bioactive molecules for healthy neurons, as well. These peculiar properties of stem cells are opening to potential therapeutic strategies for the treatment of severe neurodegenerative disorders, for which the absence of effective treatment options leads to an increasingly socio-economic burden. Currently, the introduction of new technologies in the field of stem cells and the implementation of alternative cell tissues sources are pointing to exciting frontiers in this area of research. Here, we provide an update of the current knowledge about source and administration routes of stem cells, and review light and shadows of cells replacement therapy for the treatment of the three main neurodegenerative disorders (Amyotrophic lateral sclerosis, Parkinson's, and Alzheimer's disease).

Development of a Pharmacogenetic Lab-on-Chip Assay Based on the In-Check Technology to Screen for Genetic Variations Associated to Adverse Drug Reactions to Common Chemotherapeutic Agents.

BACKGROUND: Antineoplastic agents represent the most common class of drugs causing Adverse Drug Reactions (ADRs). Mutant alleles of genes coding for drug-metabolizing enzymes are the best studied individual risk factors for these ADRs. Although the correlation between genetic polymorphisms and ADRs is well-known, pharmacogenetic tests are limited to centralized laboratories with expensive or dedicated instrumentation used by specialized personnel. Nowadays, DNA chips have overcome the major limitations in terms of sensibility, specificity or small molecular detection, allowing the simultaneous detection of several genetic polymorphisms with time and costs-effective advantages. In this work, we describe the design of a novel silicon-based lab-on-chip assay able to perform low-density and high-resolution multi-assay analysis (amplification and hybridization reactions) on the In-Check platform. METHODS: The novel lab-on-chip was used to screen 17 allelic variants of three genes associated with adverse reactions to common chemotherapeutic agents: DPYD (Dihydropyrimidine dehydrogenase), MTHFR (5,10-Methylenetetrahydrofolate reductase) and TPMT (Thiopurine S-methyltransferase). RESULTS: Inter- and intra assay variability were performed to assess the specificity and sensibility of the chip. Linear regression was used to assess the optimal hybridization temperature set at 52 °C (R2 ≈ 0.97). Limit of detection was 50 nM. CONCLUSIONS: The high performance in terms of sensibility and specificity of this lab-on-chip supports its further translation to clinical diagnostics, where it may effectively promote precision medicine.

Effect of a Bone Marrow-Derived Extracellular Matrix on Cell Adhesion and Neural Induction of Dental Pulp Stem Cells.

Extracellular matrix (ECM) represents an essential component of the cellular niche. In this conditioned microenvironment, the proliferation rates and differentiation states of stem cells are regulated by several factors. In contrast, in in vitro experimental models, cell growth, or induction procedures toward specific cell lines usually occur in contact with plastic, glass, or biogel supports. In this study, we evaluated the effect of a decellularized ECM, derived from bone marrow stem cells, on the neuronal differentiation of mesenchymal stem cells (MSCs) extracted from dental pulp (Dental Pulp Stem Cells - DPSCs). Since DPSCs derive from neuroectodermal embryonic precursors, they are thought to have a greater propensity toward neuronal differentiation than MSCs isolated from other sources. We hypothesized that the presence of a decellularized ECM scaffold could act positively on neuronal-DPSC differentiation through reproduction of an in vivo-like microenvironment. Results from scanning electron microscopy, immunofluorescence, and gene expression assays showed that ECM is able to positively influence the morphology of cells and their distribution and the expression of specific neuronal markers (i.e., NF-L, NF-M, NF-H, PAX6, MAP2).

Biocompatibility between Silicon or Silicon Carbide surface and Neural Stem Cells.

Silicon has been widely used as a material for microelectronic for more than 60 years, attracting considerable scientific interest as a promising tool for the manufacture of implantable medical devices in the context of neurodegenerative diseases. However, the use of such material involves responsibilities due to its toxicity, and researchers are pushing towards the generation of new classes of composite semiconductors, including the Silicon Carbide (3C-SiC). In the present work, we tested the biocompatibility of Silicon and 3C-SiC using an in vitro model of human neuronal stem cells derived from dental pulp (DP-NSCs) and mouse Olfactory Ensheathing Cells (OECs), a particular glial cell type showing stem cell characteristics. Specifically, we investigated the effects of 3C-SiC on neural cell morphology, viability and mitochondrial membrane potential. Data showed that both DP-NSCs and OECs, cultured on 3C-SiC, did not undergo consistent oxidative stress events and did not exhibit morphological modifications or adverse reactions in mitochondrial membrane potential. Our findings highlight the possibility to use Neural Stem Cells plated on 3C-SiC substrate as clinical tool for lesioned neural areas, paving the way for future perspectives in novel cell therapies for neuro-degenerated patients.

Ag-NPs induce apoptosis, mitochondrial damages and MT3/OSGIN2 expression changes in an in vitro model of human dental-pulp-stem-cells-derived neurons.

Silver nanoparticles (Ag-NPs) are one of the most popular nanotechnologies because of their unique antibacterial and antifungal properties. Given their increasing use in a wide range of commercial, biomedical and food products, exposure to Ag-NPs is now a reality in people's lives. However, there is a serious lack of information regarding their potential toxic effects in the central nervous system. In this study, we investigated the biocompatibility of "homemade" Ag-NPs in an in vitro model of human neurons derived from dental pulp mesenchymal stem cells. Our results showed that acute exposure to Ag-NPs cause cytotoxicity, by triggering cell apoptosis, damaging neuronal connections, affecting the mitochondrial activity and changing the mRNA expression level of MT3 and OSGIN2, two genes involved in heavy metals metabolism and cellular growth during oxidative stress conditions. Further studies are needed to understand the molecular mechanisms and the physiological consequences underlying Ag-NPs exposure.

Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disease where activated glia release pro-inflammatory cytokines that trigger a vicious cycle of neurodegeneration in the absence of resolution of inflammation. Given the well-established role of histamine as a neuron-to-glia alarm signal implicated in brain disorders, the aim of this study was to investigate the expression and regulation of the histaminergic pathway in microglial activation in ALS mouse model and in humans. By examining the contribution of the histaminergic system to ALS, we found that particularly via H1 and H4 receptors, histamine promoted an anti-inflammatory profile in microglia from SOD1-G93A mice by modulating their activation state. A decrease in NF-κB and NADPH oxidase 2 with an increase in arginase 1 and P2Y12 receptor was induced by histamine only in the ALS inflammatory environment, but not in the healthy microglia, together with an increase in IL-6, IL-10, CD163, and CD206 phenotypic markers in SOD1-G93A cells. Moreover, histaminergic H1, H2, H3, and H4 receptors, and histamine metabolizing enzymes histidine decarboxylase, histamine N-methyltransferase, and diamine oxidase were found deregulated in spinal cord, cortex, and hypothalamus of SOD1-G93A mice during disease progression. Finally, by performing a meta-analysis study, we found a modulated expression of histamine-related genes in cortex and spinal cord from sporadic ALS patients. Our findings disclose that histamine acts as anti-inflammatory agent in ALS microglia and suggest a dysregulation of the histaminergic signaling in ALS.

A neurocutaneous phenotype with paired hypo- and hyperpigmented macules, microcephaly and stunted growth as prominent features.

Neurocutaneous disorders represent a heterogeneous group of conditions affecting the skin (with pigmentary/vascular abnormalities, hamartomas or tumors) and the central and peripheral nervous systems. In recent years, besides the well-known neurocutaneous diseases (e.g., the different forms of neurofibromatosis, tuberous sclerosis complex, Sturge-Weber syndrome and mosaic pigmentary/hamartomatous disorders), new distinctive syndromes have been characterized, extending our knowledge on the spectrum of these conditions. The concurrent presence of pigmentary abnormalities (both of the hypo- and hyperpigmented type), and primary microcephaly has not been commonly reported. We report on a 4.5-year-old girl with primary microcephaly, who had in addition moderate to severe developmental delay, behavioral and stereotypic abnormalities and a cutaneous pattern of paired hypo- and hyperpigmented lesions variously distributed over the body, particularly on the trunk. Failure to thrive and mild facial dysmorphic features were also present. To our knowledge, this complex malformation (neurocutaneous) phenotype has not been previously reported.

CXCR4 and CXCL12 immunoreactivities differentiate primary non-small-cell lung cancer with or without brain metastases.

Synchronous or metachronous brain metastases (BMs) occur in about 33% of patients affected by non-small-cell lung cancer (NSCLC). To date, no reliable biological marker is able to identify patients who will develop BMs. In the present study, using a quantitative double-labeling immunofluorescence analysis, we evaluated the expression of chemokine CXCL12 and its receptor, CXCR4, in primary NSCLC histological specimens of patients with and without BMs. The immunoreactivity of CXCL12 and CXCR4 was significantly higher in NSCLC samples of patients with BMs. We performed Receiver Operating Characteristics (ROC) analysis in order to define optimal cut-off values for CXCL12 and CXCR4 immunoreactivity that could discriminate between NSCLC patients without and with BMs. ROC curves showed a good diagnostic accuracy and adequate predictive power for both CXCL12 and CXCR4. These findings suggest a possible role for the CXCL12/CXCR4 axis in the metastatic evolution of NSCLC, and its potential use as prognostic markers and drug targets.