α-Synuclein in blood exosomes immunoprecipitated using neuronal and oligodendroglial markers distinguishes Parkinson's disease from multiple system atrophy.

The diagnosis of Parkinson's disease (PD) and atypical parkinsonian syndromes is difficult due to the lack of reliable, easily accessible biomarkers. Multiple system atrophy (MSA) is a synucleinopathy whose symptoms often overlap with PD. Exosomes isolated from blood by immunoprecipitation using CNS markers provide a window into the brain's biochemistry and may assist in distinguishing between PD and MSA. Thus, we asked whether α-synuclein (α-syn) in such exosomes could distinguish among healthy individuals, patients with PD, and patients with MSA. We isolated exosomes from the serum or plasma of these three groups by immunoprecipitation using neuronal and oligodendroglial markers in two independent cohorts and measured α-syn in these exosomes using an electrochemiluminescence ELISA. In both cohorts, α-syn concentrations were significantly lower in the control group and significantly higher in the MSA group compared to the PD group. The ratio between α-syn concentrations in putative oligodendroglial exosomes compared to putative neuronal exosomes was a particularly sensitive biomarker for distinguishing between PD and MSA. Combining this ratio with the α-syn concentration itself and the total exosome concentration, a multinomial logistic model trained on the discovery cohort separated PD from MSA with an AUC = 0.902, corresponding to 89.8% sensitivity and 86.0% specificity when applied to the independent validation cohort. The data demonstrate that a minimally invasive blood test measuring α-syn in blood exosomes immunoprecipitated using CNS markers can distinguish between patients with PD and patients with MSA with high sensitivity and specificity. Future optimization and validation of the data by other groups would allow this strategy to become a viable diagnostic test for synucleinopathies.

Cross-Sectional Analysis of Exome Sequencing Diagnosis in Patients With Neurologic Phenotypes Facing Barriers to Clinical Testing.

Background and Objectives: Exome sequencing (ES) demonstrates a 20-50 percent diagnostic yield for patients with a suspected monogenic neurologic disease. Despite the proven efficacy in achieving a diagnosis for such patients, multiple barriers for obtaining exome sequencing remain. This study set out to assess the efficacy of ES in patients with primary neurologic phenotypes who were appropriate candidates for testing but had been unable to pursue clinical testing. Methods: A total of 297 patients were identified from the UCLA Clinical Neurogenomics Research Center Biobank, and ES was performed, including bioinformatic assessment of copy number variation and repeat expansions. Information regarding demographics, clinical indication for ES, and reason for not pursuing ES clinically were recorded. To assess diagnostic efficacy, variants were interpreted by a multidisciplinary team of clinicians, bioinformaticians, and genetic counselors in accordance with the American College of Medical Genetics and Genomics variant classification guidelines. We next examined the specific barriers to testing for these patients, including how frequently insurance-related barriers such as coverage denials and inadequate coverage of cost were obstacles to pursuing exome sequencing. Results: The cohort primarily consisted of patients with sporadic conditions (n = 126, 42.4%) of adult-onset (n = 239, 80.5%). Cerebellar ataxia (n = 225, 75.8%) was the most common presenting neurologic phenotype. Our study found that in this population of mostly adult patients with primary neurologic phenotypes that were unable to pursue exome sequencing clinically, 47 (15.8%) had diagnostic results while an additional 24 patients (8.1%) had uncertain results. Of the 297 patients, 206 were initially recommended for clinical exome but 88 (42.7%) could not pursue ES because of insurance barriers, of whom 14 (15.9%) had diagnostic findings, representing 29.8% of all patients with diagnostic findings. In addition, the incorporation of bioinformatic repeat expansion testing was valuable, identifying a total of 8 pathogenic repeat expansions (17.0% of all diagnostic findings) including 3 of the common spinocerebellar ataxias and 2 patients with Huntington disease. Discussion: These findings underscore the importance and value of clinical ES as a diagnostic tool for neurogenetic disease and highlight key barriers that prevent patients from receiving important clinical information with potential treatment and psychosocial implications for patients and family members.

Steric effects in peptide and protein exchange with activated disulfides.

Disulfide exchange is an important bioconjugation tool, enabling chemical modification of peptides and proteins containing free cysteines. We previously reported the synthesis of a macromer bearing an activated disulfide and its incorporation into hydrogels. Despite their ability to diffuse freely into hydrogels, larger proteins were unable to undergo in-gel disulfide exchange. In order to understand this phenomenon, we synthesized four different activated disulfide-bearing model compounds (Mn = 300 Da to 10 kDa) and quantified their rate of disulfide exchange with a small peptide (glutathione), a moderate-sized protein (ß-lactoglobulin), and a large protein (bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4, and 8.0) to mimic biological systems. Rate constants of exchange depend significantly on the size and accessibility of the thiolate. pH also significantly affects the rate of reaction, with the faster reactions occurring at higher pH. Surprisingly, little difference in exchange rates is seen between macromolecular disulfides of varying size (Mn = 2 kDa - 10 kDa), although all undergo exchange more slowly than their small molecule analogue (MW = 300 g/mol). The maximum exchange efficiencies (% disulfides exchanged after 24 h) are not siginificantly affected by thiol size or pH, but somewhat affected by disulfide size. Therefore, while all three factors investigated (pH, disulfide size, and thiolate size) can influence the exchange kinetics and extent of reaction, the size of the thiolate and its accessibility plays the most significant role.

Acute pharmacogenetic dystonic reactions in a family with the CYP2D6 *41 allele: a case report.

BACKGROUND: Dystonia is a known neurological complication of certain medications; however, the mechanism behind such effects is often undetermined. Similarly, the clinical pharmacogenomic effects associated with various alleles of the cytochrome P450 family of proteins, and their role in acute dystonic reactions, are also presently unknown. CASE PRESENTATION: We describe a woman presenting with acute dystonic reactions to ondansetron, prochlorperazine, and metoclopramide followed by persistent focal dystonia. A similar family history was reported in her siblings and her father to prochlorperazine, drugs all metabolized by the cytochrome P450 2D6 (CYP2D6) enzyme. Pharmacogenomic testing indicated the patient was heterozygous for the intermediate metabolizer *41 allele (CYP2D6 2988G>A, NM_000106.6:c.985+39G>A, rs28371725). Her father was homozygous for this CYP2D6 *41 allele, and consequently, her siblings were obligate carriers. CONCLUSIONS: The metabolism of ondansetron, metoclopramide, or prochlorperazine in patients with the *41 CYP2D6 allele has not been studied. In this family, clinical evidence implicates the *41 CYP2D6 allele as causing extrapyramidal adverse pharmacologic reactions. Patients with a family history of medication-induced dystonia involving these medications should be considered for pharmacogenomic testing, and patients carrying the *41 CYP2D6 allele should consider reduction or avoidance of CYP2D6-mediated medications to minimize the potential risk of adverse extrapyramidal effects.

Macro-architectures in spinal cord scaffold implants influence regeneration.

Biomaterial scaffold architecture has not been investigated as a tunable source of influence on spinal cord regeneration. This study compared regeneration in a transected spinal cord within various designed-macro-architecture scaffolds to determine if these architectures alone could enhance regeneration. Three-dimensional (3-D) designs were created and molds were built on a 3-D printer. Salt-leached porous poly(epsilon-caprolactone) was cast in five different macro-architectures: cylinder, tube, channel, open-path with core, and open-path without core. The two open-path designs were created in this experiment to compare different supportive aspects of architecture provided by scaffolds and their influence on regeneration. Rats received T8 transections and implanted scaffolds for 1 and 3 months. Overall morphology and orientation of sections were characterized by H&E, luxol fast blue, and cresyl violet staining. Borders between intact gray matter and non-regenerated defect were observed from GFAP immunolabeling. Nerve fibers and regenerating axons were identified with Tuj-1 immunolabeling. The open-path designs allowed extension of myelinated fibers along the length of the defect both exterior to and inside the scaffolds and maintained their original defect length up to 3 months. In contrast, the cylinder, tube, and channel implants had a doubling of defect length from secondary damage and large scar and cyst formation with no neural tissue bridging. The open-path scaffold architectures enhanced spinal cord regeneration compared to the three other designs without the use of biological factors.

Brain cortex regeneration affected by scaffold architectures.

OBJECT: The aim of this study was to compare designed scaffolds with a random-pored sponge scaffold to determine what role scaffold architecture plays in a cortical injury model. METHODS: Cylindrical scaffolds (3x3 mm) were made of a poly-(epsilon-caprolactone) polymer with 2 different molds from a 3D printer and had either: 1) unidirectional channels and microgrooves oriented longitudinally within the cylinder or 2) orthogonally intersecting channels and axial microgrooves within the cylinder. Additional randomized porosity was imparted using a salt-leaching method. A control scaffold without channels or microgrooves but containing random pores was also made. Scaffolds were implanted for 1, 4, and 8 weeks in a cylindrical defect created 3 mm posterior to the bregma in rat cortex. Control animals had tissue removed but received no implant. Brains were coronally cryosectioned and sections were stained. Antibodies for nestin, glial fibrillary acidic protein (GFAP), and TUJ1 were used to identify neural progenitors, activated astrocytes, and neuronal axons. Tissue ingrowth (H & E), astrocytic infiltration (GFAP), parenchymal inflammation (GFAP), and defect width (H & E) were quantified from images. RESULTS: Defect widths grew and parenchymal inflammation decreased over time with no statistical difference between groups. Total tissue ingrowth and astrocytic infiltration increased over time and was greatest in the orthogonal design group. Specific cell ingrowth, which was aligned with microgrooves interiorly in the orthogonal group and exteriorly in the longitudinal channel group, was qualitatively assessed from nestin and TUJ1 labeling. CONCLUSIONS: Scaffold architecture can benefit brain tissue regeneration by integrating the following design principles: 1) large (100s of micrometers) pores or channels oriented toward the parenchyma for increased astrocytic infiltration; 2) microgrooves oriented in the desired direction of cellular migration and neuronal alignment; and 3) fully interconnecting channels for cellular migration and tissue integration.

Poly(epsilon-caprolactone) and poly (L-lactic-co-glycolic acid) degradable polymer sponges attenuate astrocyte response and lesion growth in acute traumatic brain injury.

This study evaluated the response of rat brain to 2 degradable polymers (poly (L-lactic-co-glycolic acid) (PLGA), and poly(epsilon-caprolactone) (PCL)), two common materials in tissue engineering. PLGA has been extensively studied in the brain for controlled drug release as injectable microspheres and is generally accepted as biocompatible in that capacity. Biocompatibility in other forms and for different functions in the brain has not been widely studied. PCL was chosen as an alternative to PLGA for its slower degradation and less-acidic pH upon degradation. Porous scaffolds were made from both polymers and implanted into rat cerebral cortex for 1 and 4 weeks. Morphology, defect size, activation of microglia (OX-42) and astrocytes (glial fibrillary acidic protein (GFAP)), infiltration of activated macrophages (major histocompatibility complex (MHC)-II), and ingrowth of neurons (beta-tubulin type III (Tuj-1)) and progenitor cells (nestin) were analyzed using hematoxylin and eosin staining and immunofluorescence. PCL induced a lower inflammatory response than PLGA, as demonstrated by lower MHC-II and GFAP expression and greater ingrowth. Both polymers alleviated astrocytic activation and prevented enlargement of the defect. Tuj-1-, nestin-, and GFAP-positive cells were observed growing on both polymers at the peripheries of the sponge implants, demonstrating their permissiveness to neural ingrowth. These findings suggest that both polymers attenuate secondary death and scarring and that PCL might have advantages over PLGA.

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells.

Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm-400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying.

Complex dynamic substrate control: dual-tone hydrogel photoresists allow double-dissociation of topography and modulus.

Complex substrate control is demonstrated with a dual-tone hydrogel photoresist. By exposing a photodegradable hydrogel to UV light through a photomask, both swollen and eroded micropatterns with a decreased modulus can be created on the surface under different exposure conditions. This provides an important tool for investigating the synergistic effects of spatially heterogeneous mechanical and topological cues on cell behavior.

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a protein that plays a crucial role in diverse cellular functions, from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Here, we describe the stabilization of bFGF by covalent conjugation with a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors--such as heat, mild and harsh acidic conditions, storage and proteolytic degradation--unlike native bFGF. Following the application of stress, the conjugate was also significantly more active than the control conjugate system in which the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for the stabilization of heparin-binding growth factors in general.

Two-photon lithography in the future of cell-based therapeutics and regenerative medicine: a review of techniques for hydrogel patterning and controlled release.

In recent decades, there has been considerable interest in using photochemistry to produce biomaterials, owing to their ability to be used in the presence of biological material. Two-photon-induced photoreactions have been used to produce materials for optical data storage and microfabrication and, recently, researchers have exploited two-photon-induced chemical processes to create biomaterials. Researchers have used two-photon-induced lithography to fabricate hydrogels with well-defined chemical and physical properties in 3D through network polymerization, functionalization, uncaging and degradation, as described in this article. Fabrication and modification of chemical and physical architecture of biomaterials in 3D with submicron resolution will allow the elucidation of more complex relationships in cell behavior and tissue development and introduce pathways to engineering complex tissues.