Biology subjects

Kwok, J. C. F.

Publications and source records attributed to Kwok, J. C. F..

14 recordsLinked to original sources

bioRxiv2026

Intertwined Autophagy and Integrin Dynamics Shape Axon Growth and Regeneration

Autophagy is a central pathway for cellular homeostasis, mediating degradation and recycling of cytoplasmic components through lysosomal processing. Although autophagy has been implicated in axon growth and neuronal injury responses, its role in axon regeneration remains incompletely understood. In parallel, integrin trafficking and focal adhesion dynamics are critical determinants of axonal growth, and increasing evidence indicates that autophagy regulates focal adhesion turnover in non-neuronal cells; however, whether this mechanism operates in neurons remains unknown. Here, we investigated the dynamics of autophagy during axon growth and regeneration in adult sensory neurons and examined its relationship with integrin trafficking. Using live imaging, we analyzed autophagic vesicles and integrin-containing compartments in axons under basal conditions and following axotomy. We find that axonal injury induces marked short- and long-term alterations in distal axon and growth cone autophagic vesicle dynamics, integrin trafficking, and autophagy-associated integrin turnover. Importantly, these changes correlate with axonal growth and regenerative capacity. Furthermore, we identify a functional interplay between autophagy and integrins, suggesting bidirectional regulation in which autophagy contributes to adhesion receptor recycling, while integrins feedback to modulate autophagy. Finally, pharmacological modulation indicates that autophagy plays a critical role in both axonal growth and regeneration, and that rapamycin enhances regenerative responses, likely through modulation of autophagy-dependent integrin recycling rather than a general increase in autophagic activity. Together, these findings reveal a bidirectional feedback system between integrin-mediated extracellular cues and autophagy-dependent intracellular trafficking that jointly orchestrates axon growth and regeneration.

neuroscience↗

bioRxiv2026

4-Methylumbelliferone Restores Age-Related Changes in Perineuronal Nets, Memory and Neuroinflammation

Aging leads to cognitive decline due to reduced neuronal plasticity and increased brain inflammation. Disruption of perineuronal nets (PNNs) is a recognized strategy to enhance neuroplasticity. Hyaluronan forms the backbone of PNNs, and 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, has been proposed to disrupt PNNs and enhance neuroplasticity in young rodents. In addition, 4-MU is also known as an immune response regulator, although its effects within the central nervous system (CNS) have been less characterized. In this study, we investigated the impact of long-term oral 4-MU administration in aged mice (20-22 months old), focusing on PNNs intensity, memory, and neuroinflammation. PNN intensity increased with age, and 4-MU treatment reduced it in 22-month-old mice to levels observed in 10-month-old animals; in the novel object recognition test, treated 22-month-old mice performed comparably to or better than 10-month-old controls. Moreover, markers of aging-associated neuroinflammation--including astrocytic and microglial activation as well as peripheral immune cell infiltration--were normalized to 10-month-old levels or further diminished following 4-MU treatment. Importantly, chronic 4-MU administration was well tolerated in aged mice, with no serious adverse effects observed. Together, these results suggest that 4-MU mitigates PNNs accumulation and neuroinflammaging while enhancing recognition memory, supporting its potential as a safe therapeutic approach for age-related cognitive decline.

neuroscience↗

bioRxiv2026

Chondroitin 4-sulphate depletion enhances synaptic plasticity and memory in aging

Perineuronal nets (PNNs), specialised extracellular matrix structures enriched in chondroitin sulphate proteoglycans (CSPGs), are key regulators of synaptic plasticity, learning, and memory. Aging is characterised by a shift in chondroitin sulphate composition toward increased chondroitin-4-sulfation (C4S) and reduced C6S, a pattern associated with declining cognitive flexibility. Here, we investigated how selective reduction of C4S affects PNN structure, PV-interneuron connectivity, and cognitive performance across the lifespan. Conditional deletion of the C4-sulfotransferase Chst11 markedly reduced C4S levels and diminished dendritic PNN complexity while preserving somatic PNN structure. This partial destabilisation of PNNs increased excitatory synaptic input onto PV interneurons in both young and aged mice, without major alterations in basal hippocampal transmission or long-term potentiation. Behaviourally, Chst11 knockout mice showed robust and persistent protection against age-related cognitive decline. Working memory performance remained stable across aging, short-term spatial memory was enhanced from early adulthood onward, and object recognition memory was significantly prolonged at all retention delays, even in old age. Sociability and social novelty preference were also preserved longer in aging knockouts compared with controls. These improvements occurred despite an overall preservation of PNN architecture, indicating that modifying sulphation rather than removing CSPGs is sufficient to enhance plasticity. Our findings demonstrate that reducing C4S through Chst11 deletion confers long-lasting enhancements in cognitive function and mitigates aging-related decline. Targeting CS-GAG sulphation patterns may therefore represent a promising strategy for maintaining cognitive resilience and restoring plasticity in aging or neurodegenerative conditions.

neuroscience↗

bioRxiv2026

Long-Term Expression and Safety of AAV1-Mediated PI3Kδ Overexpression in the Adult Rat Cortex

Mature CNS neurons are incapable of sufficiently regenerating their axons following spinal cord injury (SCI). This is largely due to developmental changes in epigenetic control leading to suppression of axon growth transcriptomic profile, leading to a shift towards synapse function support. Recently, manipulating the PI3K/Akt/mTOR pathway through PI3K{delta} overexpression in cortical neurons enhanced axonal regeneration of corticospinal tract axons, which was accompanied by functional recovery monitored for up to 16 weeks. However, PI3K is more widely known for its role as an oncogene, and since overexpression is achieved by the use of AAVs, valid safety concerns are raised as it is unknown what the long-term consequences of sustained PI3K{delta} expression in the brain are, which may be necessary to achieve complete re-establishment of the motor pathway. In this study, AAV1-hSYN-PIK3CD was injected into the motor cortex of rats, which survived for 1 year. Comparison with uninjected control animals reveal stable PI3K{delta} expression and sustained pathway activation through increased pS6. PI3K{delta}-treated animals show absence of tumour formation, neural soma hypertrophy, glial cell activation, or haematological or biochemical abnormalities. Thus, long-term neuronal PI3K{delta} expression appears to be well tolerated and may provide a safe and durable strategy to promote functional repair following SCI.

neuroscience↗

bioRxiv2025

Exercise remodels hippocampal extracellular matrix to alleviate chondroitin-4-sulphate-induced memory impairment.

Research utilising exercise therapeutically to enhance neuroplasticity mainly reports enhanced expression of neurotrophic (neural growth) factors which are associated with increased neurogenesis, structural changes, and improved memory. However, these neural changes are tightly regulated by both promotors and inhibitors of neuroplasticity, and the effect of exercise on inhibitory pathways is currently understudied. We show that exercise also modulates inhibitors of neuroplasticity. Six weeks of treadmill training reduced the gene expression of aggrecan, a chondroitin sulphate proteoglycan (CSPG), and altered the composition of CSPG containing structures, perineuronal nets, in the rodent hippocampus. Genetically manipulating chondroitin-4-sulphation of CSPGs by overexpressing hippocampal Chst11 impaired memory performance, which was mitigated by treadmill training. We provide evidence that hippocampal CSPGs are involved in object recognition memory, and that there is a link between exercise, modulating hippocampal inhibitors of neuroplasticity, and memory performance.

neuroscience↗

bioRxiv2023

Hyperactive PI3Kinase delta enables long distance regeneration of the rat corticospinal tract

Maturation of central nervous system neurons leads to loss of their intrinsic regeneration potential. In particular after injury of the adult spinal cord there is minimal regeneration of corticospinal axons, which control gait and fine movement. Previous work has shown that knockdown of PTEN to increase PIP3 levels can promote regeneration in young animals, but the effect is much less in adults probably due to low PIP3 production. Here, we have transduced sensorimotor cortex neurons with a hyperactive form of PI3K, PI3K{delta}, which increases PIP3 in mature neurons. This enables cortical neurons to regenerate corticospinal axons and improve behavioural outcomes. We used a C4 dorsal column lesion model in adult rats and injected the right motor cortex at 4 sites concurrently with a mixture AAV1-PIK3CD and AAV1-eGFP or titre matched AAV1-eGFP only. We allowed rats to survive for 6, 9, 12 or 16 weeks. Immunostaining showed 70 - 80% co-expression in cortical neurons which remained stable at both 12 and 16 weeks. We counted GFP labelled axons in 20 m spinal cord sections. In PI3KCD-treated animals many axons were seen to have regenerated around the margins of lesions, collecting into a knot of axons with the typical appearance of regeneration at the caudal end. Tracing down the cord, and excluding axons and neurites that could have come from unlesioned ventral CST, we found axons extending up to 1 cm below lesions, numbers decreasing with distance from the lesion. After 16 weeks there were circa 200 axons at the caudal end of lesions with a regeneration index of 0.2, with half this number at 12 weeks. Behavioural testing for 16 weeks revealed functional improvements in skilled paw reaching, grip strength and ladder rung walking in rats treated with PIK3CD compared to GFP only controls. In addition to behavioural testing, functional recovery of PIK3CD treated rats was confirmed with electrophysiological recordings during which we stimulated the right pyramid. Cord dorsum potentials (CDPs) above and below lesion and EMG forepaw distal flexor muscles showed greatly increased connectivity compared with GFP only controls, lesion only controls and uninjured shams. We conclude that forcing upregulation of PI3K{delta} in cortical neurons leads to robust regeneration after spinal cord injury that results in functional restoration.

neuroscience↗

bioRxiv2023

Competitive specific anchorage of molecules onto surfaces: quantitative control of grafting densities and contamination by free anchors

The formation of surfaces decorated with biomacromolecules such as proteins, glycans or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes, and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest, and present effective guidelines towards optimizing incubation conditions for the quantitative control of surface densities. Focussing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

biophysics↗

bioRxiv2023

Alpha 9 integrin expression enables reconstruction of the spinal cord sensory pathway.

Full recovery from spinal cord injury can only occur if the axon pathways connecting the brain and spinal cord regenerate and restore motor and sensory connections. Neither sensory nor motor axons can regenerate spontaneously in the spinal cord in mammals. This failure is partly due to the lack of suitable adhesion molecules on the sensory axons that allows them to interact with the environment of the damaged spinal cord. In this rat study, an integrin adhesion molecule along with its activator was expressed in sensory neurons using an adeno-associated viral (AAV) vector. Expression of these adhesion molecules allowed sensory axons to regenerate through the spinal cord injury and all the way back to the brainstem, restoring the sensory pathway. Treated animals regained touch sensation and sensory behaviours. The integrin ligands in the injured spinal cord are tenascin-C and osteopontin, but adult PNS and CNS neurons lack receptors to them. Sensory neurons were transduced with 9 integrin, which combines with endogenous {beta}1 as 9{beta}1 (which is a tenascin/osteopontin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with 9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4.5 sensory ganglia injected with AAV1 vectors. In animals treated with 9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesion and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Regenerated axons were particularly visible at the border between white and grey matter in the dorsal cord. Stimulation of the median/sciatic nerve caused many neurons rostral to the injury to activate and express cFos. VGLUT1/2 staining indicated newly formed functional synapses above the lesion. Behavioural recovery was seen in heat, mechanical sensation and tape removal tests. Many axons regenerated from the thoracic lesions to the brainstem, a distance of 4-5 cm, equivalent to the length of 1 or 2 spinal segments in humans.

neuroscience↗

bioRxiv2023

Oral administration of 4-methylumbelliferone reduces glial scar and promotes anatomical plasticity

Following a spinal cord injury (SCI), chondroitin sulfate proteoglycans (CSPGs) are up-regulated at the glial scar inhibiting neuroregeneration. Under normal physiological condition, CSPGs interact with hyaluronan (HA) and other extracellular matrix on neuronal surface forming a macromolecular structure called perineuronal nets (PNNs) which regulate neuroplasticity. 4-methylumbelliferone (4-MU) has been used previously to down-regulate HA synthesis but not been tested in SCI. In this study, we have evaluated the effect of 4-MU, an inhibitor of HA, in a chronic contusion model of SCI in rats. At a dose of 1.2 g/kg/day of 4-MU, we observed not only the reduction of HA in the uninjured spinal cords after 60 days of 4-MU administration, but also a down-regulation of CS glycosaminoglycans (CS-GAGs). In order to assess the effect of 4-MU in chronic SCI, rats with T8 spinal contusion injury were fed with 4-MU or placebo for 8 weeks in combination with daily treadmill rehabilitation for 16 weeks to promote neuroplasticity. 4-MU treatment promoted significant sprouting of 5-hydroxytryptamine (5-HT) positive fibres into ventral horns and reduced the HA synthesis by astrocytes around the lesion site. While 4-MU reduced astrogliosis in chronic stage of SCI, the current dose was not sufficient to down-regulate the increased production of CS-GAGs or behavioural performance. Together, these data suggest that oral treatment with 4-MU is able to induce anatomical plasticity but further adjustment on the dosage will be required to benefit functional recovery after SCI.

neuroscience↗

bioRxiv2023

4-methylumbelliferone enhances neuroplasticity in the central nervous system: potential oral treatment for SCI

Perineuronal nets (PNNs) are specialised extracellular matrix (ECM) structures that act as key plasticity regulators to the central nervous system. Removal of PNNs using chondroitinase ABC injections restores plasticity, however, there are limitations to its application to due to its enzymatic nature. Here, we explore the use of a small molecule 4-methylumbelliferone (4-MU) as an alternative non-invasive strategy to reversibly remove PNNs and enhance plasticity. Oral administration of 4-MU for 10 days successfully and dynamically removed PNNs in vitro. While 4-MU, preferentially downregulated hyaluronan in the spinal cord, a down-regulation of chondroitin sulphate proteoglycans is also observed in the cortex. Long term administration for 8-weeks administration revealed a partial removal of PNNs, and that injury-induced mechanisms promoting cortical structural plasticity are linked to endogenous modulation of ECM molecules. 4-MU is a new tool to unravel the limits of normal and pathological PNN-mediated plasticity.

neuroscience↗

bioRxiv2022

Recovery of forearm and fine digit function after chronic spinal cord injury by simultaneous blockade of inhibitory matrix CSPG production and the receptor PTPσ.

Spinal cord injuries, for which there are limited effective clinical treatments, result in enduring paralysis and hypoesthesia due, in part, to the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic modulation of the potently inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) can rapidly restore robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited rapid, albeit modest, improvements in upper arm function. In the present study, we sought to further optimize and elucidate the capacity for nerve sprouting and/or regeneration to restore gross as well as fine motor control of the forearm and digits at lengthy chronic stages post injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic, non-invasive combinatorial treatment strategy designed to both reduce and overcome inhibitory CSPGs simultaneously and spatially extensively. Following a three-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment has a profound effect on functional recovery of the chronically paralyzed forelimb and paw, specifically during walking as well as precision movements of the digits. Our exciting pre-clinical findings will begin to enhance our understanding of the basic mechanisms underlying functionally beneficial regenerative events occurring at chronic injury stages for clinically relevant translational benefits. Significance statementOvercoming the persistent axon inhibitory environment following a functionally debilitating incomplete spinal cord lesion has long proven to be an elusive dilemma, especially months to years after the initial spinal injury. Current therapeutic and rehabilitative techniques for patients suffering from chronic cervical spinal insults minimally, if at all, address this structural hindrance and support limited return of crucial behaviors such as voluntary use of the arms and hands. Our investigation into the behavioral and anatomical consequences of systemically perturbing the high-affinity binding interaction between the receptor PTP{sigma} and the extracellular chondroitin sulfate proteoglycans highlight an underlying barrier to the restoration of forelimb/paw walking and eating behavior 12-weeks after a cervical spinal hemi-transection.

neuroscience↗

bioRxiv2022

A quantitative method for sizing hyaluronan and other glycosaminoglycans on surfaces

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift ({Delta}D) and the negative frequency shift (-{Delta}f) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from [~]2 kDa tetrasaccharides to [~]1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the {Delta}D/-{Delta}f ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

biophysics↗

bioRxiv2022

An in vitro neuronal model replicating the in vivo maturation and heterogeneity of perineuronal nets

The perineuronal net (PNN) is a condensed form of extracellular matrix (ECM) that enwraps specific populations of neurons and regulates plasticity. To create a PNN, only three classes of components are needed: membrane bound hyaluronan by its synthetic enzyme hyaluronan synthases (HASs), a link protein and a CSPG. However, there is redundancy within the classes as multiple HAS isoforms, link proteins and CSPGs have been found in the PNN in vivo. The effect of this heterogeneity has on PNN function is unresolved. Currently, the most common way to address this question is through the creation and study of PNN component in knockout animals. Here, we reported the development of a primary neuronal culture model which reproduces the in vivo maturation and heterogeneity of PNNs. This model accurately replicated mature cortical PNNs, both in terms of the heterogeneity in PNN composition and its maturation. PNNs transitioned from an immature punctate morphology to the reticular morphology as observed in the mature CNS. We also observed a small population of PNNs that were mature at an earlier time point and a distinct composition, highlighting further heterogeneity. This model will provide a valuable tool for the study of PNN biology, their roles in diseases and the development of PNN focused plasticity treatment.

neuroscience↗

bioRxiv2019

Semaphorin 3A binding to chondroitin sulfate E enhances the biological activity of the protein, and cross-links and rigidifies glycosaminoglycan matrices

Semaphorin 3A (Sema3A) is a secreted protein that signals to cells through binding to neuropilin and plexin receptors and provides neurons with guidance cues key for axon pathfinding, and also controls cell migration in several other biological systems. Sema3A interacts with glycosaminoglycans (GAGs), an interaction that could localize the protein within tissues and involves the C-terminal domain of the protein. This domain comprises several furin cleavage sites that are processed during secretion and in previous works have hampered recombinant production of full-length wild type Sema3A, and the biochemical analysis of Sema3A interaction with GAGs. In this work, we have developed a strategy to purify the full-length protein in high yield and identified two sequences in the C-terminal domain, KRDRKQRRQR and KKGRNRR, which confer to the protein sub nM affinity for chondroitin sulfate and heparan sulfate polysaccharides. Using chemically defined oligosaccharides and solid phase binding assays, we report that Sema3A recognizes a (GlcA-GalNAc4S6S)2 motif but not a (GlcA2S-GalNAc6S)2 motif and is thus highly specific for type E chondroitin sulfate. Functionally, we found that Sema3A rigidified CS-E films that mimic the GAG presentation within extracellular matrices (ECMs), suggesting that Sema3A may have a previously unidentified function to cross-link and thus stabilize GAG-rich ECMs. Finally, we demonstrated that the full-length Sema3A is more potent at inhibiting neurite outgrowth than the truncated or mutant forms that were previously purified and that the GAG binding sites are required to achieve full activity. The results suggest that Sema3A can rigidify and cross-link GAG matrices, implicating Sema3A could function as an extracellular matrix organizer in addition to binding to and signaling through its cognate cell surface receptors.

biochemistry↗