Redesigned chondroitinase ABC degrades inhibitory chondroitin sulfate proteoglycans in vitro and in vivo in the stroke-injured rat brain.

Injuries to the central nervous system, such as stroke and traumatic spinal cord injury, result in an aggregate scar that both limits tissue degeneration and inhibits tissue regeneration. The aggregate scar includes chondroitin sulfate proteoglycans (CSPGs), which impede cell migration and axonal outgrowth. Chondroitinase ABC (ChASE) is a potent yet fragile enzyme that degrades CSPGs, and thus may enable tissue regeneration. ChASE37, with 37-point mutations to the native enzyme, has been shown to be more stable than ChASE, but its efficacy has never been tested. To answer this question, we investigated the efficacy of ChASE37 first in vitro using human cell-based assays and then in vivo in a rodent model of stroke. We demonstrated ChASE37 degradation of CSPGs in vitro and the consequent cell adhesion and axonal sprouting now possible using human induced pluripotent stem cell (hiPSC)-derived neurons. To enable prolonged release of ChASE37 to injured tissue, we expressed it as a fusion protein with a Src homology 3 (SH3) domain and modified an injectable, carboxymethylcellulose (CMC) hydrogel with SH3-binding peptides (CMC-bp) using inverse electron-demand Diels-Alder chemistry. We injected this affinity release CMC-bp/SH3-ChASE37 hydrogel epicortically to endothelin-1 stroke-injured rats and confirmed bioactivity via degradation of CSPGs and axonal sprouting in and around the lesion. With CSPG degradation shown both in vitro by greater cell interaction and in vivo with local delivery from a sustained release formulation, we lay the foundation to test the potential of ChASE37 and its delivery by local affinity release for tissue regeneration after stroke.

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes.

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

Identification and characterization of a chondroitinase ABC with a novel carbohydrate-binding module.

Chondroitinases play important roles in structural and functional studies of chondroitin sulfates. Carbohydrate-binding module (CBM) is generally considered as an accessory module in carbohydrate-active enzymes, which promotes the association of the appended enzyme with the substrate and potentiates the catalytic activity. However, the role of natural CBM in chondroitinases has not been investigated. Herein, a novel chondroitinase ChABC29So containing an unknown domain with a predicted ß-sandwich fold was discovered from Segatella oris. Recombinant ChABC29So showed enzyme activity towards chondroitin sulfates and hyaluronic acid and acted in a random endo-acting manner. The unknown domain exhibited a chondroitin sulfate-binding capacity and was identified as a CBM. Biochemical characterization of ChABC29So and the CBM-truncated enzyme revealed that the CBM enhances the catalytic activity, thermostability, and disaccharide proportion in the final enzymatic products of ChABC29So. These findings demonstrate the role of the natural CBM in a chondroitinase and will guide future modification of chondroitinases.

Temporal Enzymatic Treatment to Enhance the Remodeling of Multiple Cartilage Microtissues into a Structurally Organized Tissue.

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development.

A systematic review and meta-analysis of chondroitinase ABC promotes functional recovery in rat models of spinal cord injury.

BACKGROUND: To comprehensively assess the neurologic recovery potential of chondroitinase ABC (ChABC) in rats after spinal cord injury (SCI). METHODS: The PubMed, Embase, ScienceDirect, Web of Science, and China National Knowledge Infrastructure databases were searched for animal experiments that evaluated the use of ChABC in the treatment of SCI up to November 2022. Studies reporting neurological function using the Basso, Beattie, and Bresnahan (BBB) scale, as well as assessments of cavity area, lesion area, and glial fibrillary acidic protein (GFAP) levels, were included in the analysis. RESULTS: A total of 46 studies were ultimately selected for inclusion. The results of the study showed that rats with SCI that received ChABC therapy exhibited a significant improvement in locomotor function after 7 days compared with controls (32 studies, weighted mean difference (WMD) = 0.58, [0.33, 0.83], p < 0.00001). Furthermore, the benefits of ChABC therapy were maintained for up to 28 days according to BBB scale. The lesion area was reduced by ChABC (5 studies, WMD = -20.94, [-28.42, -13.46], p < 0.00001). Meanwhile, GFAP levels were reduced in the ChABC treatment group (8 studies, WMD = -29.15, [-41.57, -16.72], p < 0.00001). Cavity area is not statistically significant. The subgroup analysis recommended that a single injection of 10 µL (8 studies, WMD = 2.82, [1.99, 3.65], p < 0.00001) or 20 U/mL (4 studies, WMD = 2.21, [0.73, 3.70], p = 0.003) had a better effect on improving the function. The funnel plot of the BBB scale was found to be essentially symmetrical, indicating a low risk of publication bias. CONCLUSIONS: This systematic review and meta-analysis has indicated that ChABC could improve functional recovery in rats after SCI.

Intradiscal Condoliase Injection Therapy for Recurrent Lumbar Disc Herniation: Case Series and Literature Review.

Background and objectives: Although chemonucleolysis with condoliase for lumbar disc herniation (LDH) has become common, few reports have described its application in the treatment of recurrent LDH. Therefore, this study aimed to evaluate the safety and efficacy of condoliase treatment in six patients with recurrent LDH and review the available literature on condoliase treatment for LDH. Materials and Methods: Six patients (four men and two women; mean age, 64.7 years) with recurrent LDH who were treated with condoliase at our hospital between 2019 and 2022 were included. The clinical records and images of the patients were retrospectively evaluated. In addition, the available English literature on condoliase treatment for LDH was retrieved and reviewed. Results: Among the six patients included in the study, three showed >50% improvement in leg pain after treatment, which is a lower efficacy rate than that in previous reports. In addition, two patients required surgery after treatment, which is a higher rate than that in previous reports. The mean intervertebral disc height significantly decreased from 8.4 mm before treatment to 6.9 mm after treatment, consistent with the results of previous studies. None of the cases showed Modic type I changes on magnetic resonance imaging. Conclusions: Although the efficacy of condoliase treatment for recurrent LDH may be lower than that for primary LDH, this treatment was found to be safe and applicable for recurrent LDH.

Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue.

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

Degradation of Perineuronal Nets in the Cerebellar Interpositus Nucleus Ameliorated Social Deficits in Shank3-deficient Mice.

Perineuronal nets (PNNs) are structures that contain extracellular matrix chondroitin sulfate proteoglycan and surround the soma and dendrites of various neuronal cell types. They are involved in synaptic plasticity and undertake important physiological functions. Altered expression of PNNs has been demonstrated in the brains of autism-related animal models. However, the underlying mechanism is still unknown. In this study, we demonstrated that the PNNs in the cerebellum are involved in modulating social and repetitive/inflexible behaviors in Shank3B-/- mice, an established animal model of autism spectrum disorder. First, we performed wisteria floribunda agglutinin staining of the whole brain of Shank3B-/- mice, and found wisteria floribunda agglutinin-positive PNNs are significantly increased in the cerebellar interpositus nucleus (IntP) in Shank3B-/- mice compared to control littermates. After degradation of PNNs in the IntP by chondroitinase ABC, the repetitive behaviors of Shank3B-/- mice were decreased, while their social behaviors were ameliorated. These results suggested that PNNs homeostasis is involved in the regulation of social behavior, revealing a potential therapeutic strategy targeting PNNs in the IntP for the treatment of autism spectrum disorder.

Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I.

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius's analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants.

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives.

Chondroitin sulfate (CS) has a wide range of physiological functions and clinical applications. However, the biosynthesis of chondroitin oligosaccharides (o-CHs) and sulfate derivatives with specific length is always challenging. Herein, we report enzymatic strategies for producing homogeneous o-CHs and its sulfate derivatives from microbial sourced chondroitin. Chondroitin disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides with defined structure were produced by controllably depolymerizing microbial sourced chondroitin with an engineered chondroitinase ABC I. The highest conversion rates of the above corresponding o-CHs were 65.5%, 32.1%, 12.7%, 7.2%, and 16.3%, respectively. A new efficient enzymatic sulfation system that directly initiates from adenosine 5'-triphosphate (ATP) and sulfate was developed and improved the sulfation of chondroitin from 8.3% to 85.8% by optimizing the temperature, sulfate and ATP concentration. o-CHs decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide and disaccharide were modified and the corresponding sulfate derivatives with one sulfate group were prepared. The enzymatic approaches constructed here for preparing o-CHs and its sulfate derivatives pave the way for the study of structure-activity relationship and applications.

Chronic muscle recordings reveal recovery of forelimb function in spinal injured female rats after cortical epidural stimulation combined with rehabilitation and chondroitinase ABC.

Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.

Role of Perineuronal nets in the cerebellar cortex in cocaine-induced conditioned preference, extinction, and reinstatement.

Perineuronal nets (PNNs) are cartilage-like structures of extracellular matrix molecules that enwrap in a net-like manner the cell-body and proximal dendrites of special subsets of neurons. PNNs stabilize their incoming connections and restrict plasticity. Consequently, they have been proposed as a candidate mechanism for drug-induced learning and memory. In the cerebellum, PNNs surround Golgi inhibitory interneurons and both inhibitory and excitatory neurons in the deep cerebellar nuclei (DCN). Previous studies from the lab showed that cocaine-induced conditioned memory increased PNN expression in the granule cell layer of the posterior vermis. The present research aimed to investigate the role of cerebellar PNNs in cocaine-induced conditioned preference. For this purpose, we use the enzyme chondroitinase ABC (ChABC) to digest PNNs at different time points of the learning process to ascertain whether their removal can affect drug-induced memory. Our results show that PNN digestion using ChABC in the posterior vermis (Lobule VIII) did not affect the acquisition of cocaine-induced conditioned preference. However, the removal of PNNs in Lobule VIII -but not in the DCN- disrupted short-term memory of conditioned preference. Moreover, although PNN digestion facilitated the formation of extinction, reinstatement of cocaine-induced conditioned preference was encouraged under PNN digestion. The present findings suggests that PNNs around Golgi interneurons are needed to maintain cocaine-induced Pavlovian memory but also to stabilize extinction memory. Conversely, PNN degradation within the DCN did not affect stability of cocaine-induced memories. Therefore, degradation of PNNs in the vermis might be used as a promising tool to manipulate drug-induced memory.

Transplantation of encapsulated autologous olfactory ensheathing cell populations expressing chondroitinase for spinal cord injury: A safety and feasibility study in companion dogs.

Spinal cord injury (SCI) can cause irreversible paralysis, with no regenerative treatment clinically available. Dogs with natural SCI present an established model and can facilitate translation of experimental findings in rodents to people. We conducted a prospective, single arm clinical safety study in companion dogs with chronic SCI to characterize the feasibility of intraspinal transplantation of hydrogel-encapsulated autologous mucosal olfactory ensheathing cell (mOEC) populations expressing chondroitinase ABC (chABC). mOECs and chABC are both promising therapies for SCI, and mOECs expressing chABC drive greater voluntary motor recovery than mOECs alone after SCI in rats. Canine mOECs encapsulated in collagen hydrogel can be matched in stiffness to canine SCI. Four dogs with complete and chronic loss of function caudal to a thoraco-lumbar lesion were recruited. After baseline measures, olfactory mucosal biopsy was performed and autologous mOECs cultured and transduced to express chABC, then hydrogel-encapsulated and percutaneously injected into the spinal cord. Dogs were monitored for 6 months with repeat clinical examinations, spinal MRI, kinematic gait and von Frey assessment. No adverse effects or significant changes on neurological examination were detected. MRI revealed large and variable lesions, with no spinal cord compression or ischemia visible after hydrogel transplantation. Owners reported increased pelvic-limb reflexes with one dog able to take 2-3 unsupported steps, but gait-scoring and kinematic analysis showed no significant improvements. This novel combination approach to regeneration after SCI is therefore feasible and safe in paraplegic dogs in a clinical setting. A randomised-controlled trial in this translational model is proposed to test efficacy.

Comparisons between needle puncture and chondroitinase ABC to induce intervertebral disc degeneration in rabbits.

PURPOSE: This study aimed to compare the effect of needle puncture and chondroitinase ABC (ChABC) injection on inducing intervertebral disc (IVD) degeneration (IVDD) in rabbits. METHODS: Sixteen New Zealand white rabbits were used in this study. Briefly, the rabbits were divided into four groups. In the annulus fibrosis (AF) needle puncture group, a 16-G needle was used to puncture the L5-6 and L6-7 IVDs, while in the sham group, these IVDs were not punctured. In the ChABC group, 30 µL 0.5 Unit/mL ChABC was injected into L5-6 and L6-7 IVDs using a 26-G needle, while in the vehicle group, these IVDs were injected with 30 µL phosphate-buffered saline (PBS). X-ray and MRI scans were performed at the 4th, 12th and 16th weeks postoperatively. Histological, immunohistochemical and biochemical analyses were performed at the 16th week postoperatively. RESULTS: Both needle puncture and ChABC successfully established IVDD in rabbits at 4th, 12th and 16th weeks, confirmed by X-ray and MRI scan. The progression of IVDD went in a time-dependent manner. The IVDD in the ChABC group was less severe than in the needle puncture group throughout the study. Aggrecan and type II collagen significantly decreased, while tumor necrosis factor-α and superoxide dismutase 2 increased in the needle puncture and ChABC groups, compared with the sham and PBS groups. CONCLUSIONS: Both AF needle puncture and ChABC injection can successfully induce IVDD in rabbits. Compared with ChABC injection, AF needle puncture can induce more severe IVDD.

Removal of perineuronal nets leads to altered neuronal excitability and synaptic transmission in the visual cortex with distinct time courses.

Parvalbumin-expressing (PV) interneurons fast inhibit excitatory neurons in various brain areas. Perineuronal nets (PNNs), accumulating around PV neurons, have been shown to play critical roles in neuronal function and plasticity. The cellular mechanisms underlying their functions are still in debate, for example, do PNNs contribute significantly to the excitability of inhibitory neurons especially those containing PV? On the other hand, whether PNNs have significant contributions to synaptic transmission of PV neurons is much less unknown. In this study, we designed experiments to address these questions and found that removing PNNs in vivo using chondroitinase ABC (ChABC) led to distinct changes in neuronal excitability and synaptic transmission, depending on the duration of ChABC treatment. The results showed 7 days after ChABC treatment reduced both intrinsic excitability of PV neurons and synaptic transmission to both PV neurons and excitatory neurons in the primary visual cortex. However, 1 day after ChABC treatment digested PNNs effectively but had no effects on intrinsic excitability and synaptic transmission. These results suggest the contribution of PNNs to neuronal excitability and synaptic transmission depends on different time courses of ChABC digestion.

Comparing similar versions of a connecting helix on the structure of Chondroitinase ABC I.

Regarding the existence of similar helices on the structure of different proteins, recently, novel variants of Chondroitinase ABC I (cABC I) have been constructed, where a representative helix between two structural motifs in Chondroitinase ABC I from Proteus vulgaris has been replaced by similar versions of helices found in other proteins. The previous study has revealed that the structural features and the activity of double mutants M886A/G887E (inspired by the 30 S ribosomal protein S1 from Geminocystis herdmanii) and M889I/Q891K (inspired by the chondroitin lyase from Proteus mirabilis) is comparable with that of wild-type (WT) cABC I. Here, the kinetic parameters of the enzyme activity for the WT and double mutants were determined. Of the recombinant double mutants, M889I/Q891K gave the highest catalytic efficiency with the kcat/Km value of approximately 2.3-fold increase, as compared with the WT and M886A/G887E. Modeling of experimental data showed that the mechanism of the heat-induced structural alteration, and the enzyme-substrate complex formation, changed upon mutation. These natural versions of the connecting helix can be used as an efficient linker in protein engineering studies as well as those investigations involving the use of biological linkers.

A Progressive Build-up of Perineuronal Nets in the Somatosensory Cortex Is Associated with the Development of Chronic Pain in Mice.

Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.

Aberrant Gamma-Band Oscillations in Mice with Vitamin D Deficiency: Implications on Schizophrenia and its Cognitive Symptoms.

Vitamin D plays an essential role in cognitive functions as well as regulating calcium homeostasis and the immune system. Many epidemiological studies have also shown the close relationship between vitamin D deficiency (VDD) and the risk of schizophrenia. Cortical gamma-band oscillations (GBO) are associated with cognitive functions, such as attention and memory. Patients with schizophrenia show abnormal GBO with increased spontaneous GBO and decreased evoked GBO. However, the direct effect of VDD on GBO remains unknown. Parvalbumin interneurons, which predominantly contribute to the generation of GBO, are surrounded by perineuronal nets (PNN). We sought to investigate the associations among VDD, PNN, and GBO. Here, we injected a viral vector (AAV5-DIO-ChR2-eYFP) into the basal forebrain stereotaxically and implanted electrodes for electroencephalogram (EEG). At baseline, the evoked and spontaneous EEG power at the gamma frequency band was measured in 4-month-old male PV-Cre mice. After six and twenty weeks of vitamin D deficient food administration, the power of GBO was measured in the VDD condition. Next, we injected the chondroitinase ABC (ChABC) enzyme into the frontal cortex to eliminate PNN. We found that the VDD group showed decreased power of both optogenetically- and auditory-evoked GBO, whereas the spontaneous GBO increased. Enzymatic digestion of PNN showed similar changes in GBO. Taken together, we suggest that VDD could result in decreased PNN and, consequently, increase the spontaneous GBO and decrease the evoked GBO, reminiscent of the aberrant GBO in schizophrenia. These results show that VDD might increase the risk of schizophrenia and aggravate the cognitive symptoms of schizophrenia.

Machine-Assisted Discovery of Chondroitinase ABC Complexes toward Sustained Neural Regeneration.

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, degrades the glycosaminoglycan (GAG) side chains of CSPGs and promotes tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37 °C under dilute conditions. To overcome this limitation, the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature is reported. The copolymer designs, which are based on chain length and composition of the copolymers, are identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian process regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24 h at 37 °C. Significant improvements in REA in three iterations of active learning are demonstrated while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promotes residual ChABC activity near 30%, even after one week and notably outperforms other common stabilization methods for ChABC. Together, these results highlight a promising pathway toward sustained tissue regeneration.

Extracellular Matrix and Oxidative Stress Following Traumatic Spinal Cord Injury: Physiological and Pathophysiological Roles and Opportunities for Therapeutic Intervention.

Significance: Traumatic spinal cord injury (SCI) causes significant disruption to neuronal, glial, vascular, and extracellular elements. The spinal cord extracellular matrix (ECM) comprises structural and communication proteins that are involved in reparative and regenerative processes after SCI. In the healthy spinal cord, the ECM helps maintain spinal cord homeostasis. After SCI, the damaged ECM limits plasticity and contributes to inflammation through the expression of damage-associated molecules such as proteoglycans. Recent Advances: Considerable insights have been gained by characterizing the origins of the gliotic and fibrotic scars, which not only reduce the spread of injury but also limit neuroregeneration. These properties likely limit the success of therapies used to treat patients with SCI. The ECM, which is a major contributor to the scars and normal physiological functions of the spinal cord, represents an exciting therapeutic target to enhance recovery post-SCI. Critical Issue: Various ECM-based preclinical therapies have been developed. These include disrupting scar components, inhibiting activity of ECM metalloproteinases, and maintaining iron homeostasis. Biomaterials have also been explored. However, the majority of these treatments have not experienced successful clinical translation. This could be due to the ECM and scars' polarizing roles. Future Directions: This review surveys the complexity involved in spinal ECM modifications, discusses new ECM-based combinatorial strategies, and explores the biomaterials evaluated in clinical trials, which hope to introduce new treatments that enhance recovery after SCI. These topics will incorporate oxidative species, which are both beneficial and harmful in reparative and regenerative processes after SCI, and not often assessed in pertinent literature. Antioxid. Redox Signal. 37, 184-207.