Engineering polysialic acid on Schwann cells using polysialyltransferase gene transfer or purified enzyme exposure for spinal cord injury transplantation.

Polysialic acid (PolySia) is a critical post-translational modification on the neural cell adhesion molecule (NCAM, a.k.a., CD56), important for cell migration and axon growth during nervous system development, plasticity and repair. PolySia induction on Schwann cells (SCs) enhances their migration, axon growth support and ability to improve functional recovery after spinal cord injury (SCI) transplantation. In the current investigation two methods of PolySia induction on SCs, lentiviral vector transduction of the mouse polysialytransferase gene ST8SIA4 (LV-PST) or enzymatic engineering with a recombinant bacterial PST (PSTNm), were examined comparatively for their effects on PolySia induction, SC migration, the innate immune response and axon growth after acute SCI. PSTNm produced significant PolySia induction and a greater diversity of surface molecule polysialylation on SCs as evidenced by immunoblot. In the scratch wound assay, PSTNm was superior to LV-PST in the promotion of SC migration and gap closure. At 24 h after SCI transplantation, PolySia induction on SCs was most pronounced with LV-PST. Co-delivery of PSTNm with SCs, but not transient cell exposure, led to broader induction of PolySia within the injured spinal cord due to polysialylation upon both host cells and transplanted SCs. The innate immune response after SCI, measured by CD68 immunoreactivity, was similar among PolySia induction methods. LV-PST or PSTNm co-delivery with SCs provided a similar enhancement of SC migration and axon growth support above that of unmodified SCs. These studies demonstrate that LV-PST and PSTNm provide comparable acute effects on SC polysialation, the immune response and neurorepair after SCI.

Infantile Neuroaxonal Dystrophy: Diagnosis and Possible Treatments.

Infantile Neuroaxonal Dystrophy (INAD) is a rare neurodegenerative disease that often cuts short the life span of a child to 10 years. With a typical onset at 6 months of age, INAD is characterized by regression of acquired motor skills, delayed motor coordination and eventual loss of voluntary muscle control. Biallelic mutations in the PLA2G6 gene have been identified as the most frequent cause of INAD. We highlight the salient features of INAD molecular pathology and the progress made in molecular diagnostics. We reiterate that enhanced molecular diagnostic methodologies such as targeted gene panel testing, exome sequencing, and whole genome sequencing can help ascertain a molecular diagnosis. We describe how the defective catalytic activity of the PLA2G6 gene could be potentially overcome by enzyme replacement or gene correction, giving examples and challenges specific to INAD. This is expected to encourage steps toward developing and testing emerging therapies that might alleviate INAD progression and help realize objectives of patient formed organizations such as the INADcure Foundation.

Phosphodiesterase Inhibitors as a Therapeutic Approach to Neuroprotection and Repair.

A wide diversity of perturbations of the central nervous system (CNS) result in structural damage to the neuroarchitecture and cellular defects, which in turn are accompanied by neurological dysfunction and abortive endogenous neurorepair. Altering intracellular signaling pathways involved in inflammation and immune regulation, neural cell death, axon plasticity and remyelination has shown therapeutic benefit in experimental models of neurological disease and trauma. The second messengers, cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP), are two such intracellular signaling targets, the elevation of which has produced beneficial cellular effects within a range of CNS pathologies. The only known negative regulators of cyclic nucleotides are a family of enzymes called phosphodiesterases (PDEs) that hydrolyze cyclic nucleotides into adenosine monophosphate (AMP) or guanylate monophosphate (GMP). Herein, we discuss the structure and physiological function as well as the roles PDEs play in pathological processes of the diseased or injured CNS. Further we review the approaches that have been employed therapeutically in experimental paradigms to block PDE expression or activity and in turn elevate cyclic nucleotide levels to mediate neuroprotection or neurorepair as well as discuss both the translational pathway and current limitations in moving new PDE-targeted therapies to the clinic.

Regulating Axonal Responses to Injury: The Intersection between Signaling Pathways Involved in Axon Myelination and The Inhibition of Axon Regeneration.

Following spinal cord injury (SCI), a multitude of intrinsic and extrinsic factors adversely affect the gene programs that govern the expression of regeneration-associated genes (RAGs) and the production of a diversity of extracellular matrix molecules (ECM). Insufficient RAG expression in the injured neuron and the presence of inhibitory ECM at the lesion, leads to structural alterations in the axon that perturb the growth machinery, or form an extraneous barrier to axonal regeneration, respectively. Here, the role of myelin, both intact and debris, in antagonizing axon regeneration has been the focus of numerous investigations. These studies have employed antagonizing antibodies and knockout animals to examine how the growth cone of the re-growing axon responds to the presence of myelin and myelin-associated inhibitors (MAIs) within the lesion environment and caudal spinal cord. However, less attention has been placed on how the myelination of the axon after SCI, whether by endogenous glia or exogenously implanted glia, may alter axon regeneration. Here, we examine the intersection between intracellular signaling pathways in neurons and glia that are involved in axon myelination and axon growth, to provide greater insight into how interrogating this complex network of molecular interactions may lead to new therapeutics targeting SCI.

Neuronatin-mediated aberrant calcium signaling and endoplasmic reticulum stress underlie neuropathology in Lafora disease.

Lafora disease (LD) is a teenage-onset inherited progressive myoclonus epilepsy characterized by the accumulations of intracellular inclusions called Lafora bodies and caused by mutations in protein phosphatase laforin or ubiquitin ligase malin. But how the loss of function of either laforin or malin causes disease pathogenesis is poorly understood. Recently, neuronatin was identified as a novel substrate of malin that regulates glycogen synthesis. Here we demonstrate that the level of neuronatin is significantly up-regulated in the skin biopsy sample of LD patients having mutations in both malin and laforin. Neuronatin is highly expressed in human fetal brain with gradual decrease in expression in developing and adult brain. However, in adult brain, neuronatin is predominantly expressed in parvalbumin-positive GABAergic interneurons and localized in their processes. The level of neuronatin is increased and accumulated as insoluble aggregates in the cortical area of LD brain biopsy samples, and there is also a dramatic loss of parvalbumin-positive GABAergic interneurons. Ectopic expression of neuronatin in cultured neuronal cells results in increased intracellular Ca(2+), endoplasmic reticulum stress, proteasomal dysfunction, and cell death that can be partially rescued by malin. These findings suggest that the neuronatin-induced aberrant Ca(2+) signaling and endoplasmic reticulum stress might underlie LD pathogenesis.

Lafora disease ubiquitin ligase malin promotes proteasomal degradation of neuronatin and regulates glycogen synthesis.

Lafora disease (LD) is the inherited progressive myoclonus epilepsy caused by mutations in either EPM2A gene, encoding the protein phosphatase laforin or the NHLRC1 gene, encoding the ubiquitin ligase malin. Since malin is an ubiquitin ligase and its mutations cause LD, it is hypothesized that improper clearance of its substrates might lead to LD pathogenesis. Here, we demonstrate for the first time that neuronatin is a novel substrate of malin. Malin interacts with neuronatin and enhances its degradation through proteasome. Interestingly, neuronatin is an aggregate prone protein, forms aggresome upon inhibition of cellular proteasome function and malin recruited to those aggresomes. Neuronatin is found to stimulate the glycogen synthesis through the activation of glycogen synthase and malin prevents neuronatin-induced glycogen synthesis. Several LD-associated mutants of malin are ineffective in the degradation of neuronatin and suppression of neuronatin-induced glycogen synthesis. Finally, we demonstrate the increased levels of neuronatin in the skin biopsy sample of LD patients. Overall, our results indicate that malin negatively regulates neuronatin and its loss of function in LD results in increased accumulation of neuronatin, which might be implicated in the formation of Lafora body or other aspect of disease pathogenesis.

Sequestration of chaperones and proteasome into Lafora bodies and proteasomal dysfunction induced by Lafora disease-associated mutations of malin.

Lafora disease (LD) is an autosomal recessive progressive myoclonic epilepsy characterized by the presence of intracellular polyglucosan inclusions commonly known as Lafora bodies in many tissues, including the brain, liver and skin. The disease is caused by mutations in either EPM2A gene, encoding the protein phosphatase, laforin, or EPM2B gene, encoding the ubiquitin ligase, malin. But how mutations in these two genes cause disease pathogenesis is poorly understood. In this study, we show that the Lafora bodies in the axillary skin and brain stain positively for the ubiquitin, the 20S proteasome and the molecular chaperones Hsp70/Hsc70. Interestingly, mutant malins that are misfolded also frequently colocalizes with Lafora bodies in the skin biopsy sample of the respective LD patient. The expression of disease-causing mutations of malin in Cos-7 cells results in the formation of the profuse cytoplasmic aggregates that colocalize with the Hsp70/Hsc70 chaperones and the 20S proteasome. The mutant malin expressing cells also exhibit proteasomal dysfunction and cell death. Overexpression of Hsp70 decreases the frequency of the mutant malin aggregation and protects from mutant malin-induced cell death. These findings suggest that Lafora bodies consist of abnormal proteins, including mutant malin, targeted by the chaperones or the proteasome for their refolding or clearance, and failure of these quality control systems could lead to LD pathogenesis. Our data also indicate that the Hsp70 chaperone could be a potential therapeutic target of LD.

Co-chaperone CHIP stabilizes aggregate-prone malin, a ubiquitin ligase mutated in Lafora disease.

Lafora disease (LD) is an autosomal recessive neurodegenerative disorder caused by mutation in either the dual specificity phosphatase laforin or ubiquitin ligase malin. A pathological hallmark of LD is the accumulation of cytoplasmic polyglucosan inclusions commonly known as Lafora bodies in both neuronal and non-neuronal tissues. How mutations in these two proteins cause disease pathogenesis is not well understood. Malin interacts with laforin and recruits to aggresomes upon proteasome inhibition and was shown to degrade misfolded proteins. Here we report that malin is spontaneously misfolded and tends to be aggregated, degraded by proteasomes, and forms not only aggresomes but also other cytoplasmic and nuclear aggregates in all transfected cells upon proteasomal inhibition. Malin also interacts with Hsp70. Several disease-causing mutants of malin are comparatively more unstable than wild type and form aggregates in most transfected cells even without the inhibition of proteasome function. These cytoplasmic and nuclear aggregates are immunoreactive to ubiquitin and 20 S proteasome. Interestingly, progressive proteasomal dysfunction and cell death is also most frequently observed in the mutant malin-overexpressed cells compared with the wild-type counterpart. Finally, we demonstrate that the co-chaperone carboxyl terminus of the Hsc70-interacting protein (CHIP) stabilizes malin by modulating the activity of Hsp70. All together, our results suggest that malin is unstable, and the aggregate-prone protein and co-chaperone CHIP can modulate its stability.

Induction of chemokines, MCP-1, and KC in the mutant huntingtin expressing neuronal cells because of proteasomal dysfunction.

Huntington's disease is a hereditary neurodegenerative disorder caused by an aberrant polyglutamine expansion in the amino terminus of the huntingtin protein. The resultant mutant huntingtin form aggregates in neurons and causes neuronal dysfunction and degeneration in many ways including transcriptional dysregulation. Here, we report that the expression of mutant huntingtin in the mouse neuroblastoma cell results in massive transcriptional induction of several chemokines including monocyte chemoattractant protein-1 (MCP-1) and murine chemokine (KC). The mutant huntingtin expressing cells also exhibit proteasomal dysfunction and down-regulation of NF-kappaB activity in a time-dependent manner and both these phenomena regulate the expression of MCP-1 and KC. The expression of MCP-1 and KC are increased in the mutant huntingtin expressing cells in response to mild proteasome inhibition. However, the expression of MCP-1 and KC and proteasome activity are not altered and inflammation is rarely observed in the brain of 12-week-old Huntington's disease transgenic mice in comparison with their age-matched controls. Our result suggests that the mutant huntingtin-induced proteasomal dysfunction can up-regulate the expression of MCP-1 and KC in the neuronal cells and therefore might trigger the inflammation process.