Brain Targeted AAV1-GALC Gene Therapy Reduces Psychosine and Extends Lifespan in a Mouse Model of Krabbe Disease.

Krabbe disease (KD) is a progressive and devasting neurological disorder that leads to the toxic accumulation of psychosine in the white matter of the central nervous system (CNS). The condition is inherited via biallelic, loss-of-function mutations in the galactosylceramidase (GALC) gene. To rescue GALC gene function in the CNS of the twitcher mouse model of KD, an adeno-associated virus serotype 1 vector expressing murine GALC under control of a chicken ß-actin promoter (AAV1-GALC) was administered to newborn mice by unilateral intracerebroventricular injection. AAV1-GALC treatment significantly improved body weight gain and survival of the twitcher mice (n = 8) when compared with untreated controls (n = 5). The maximum weight gain after postnatal day 10 was significantly increased from 81% to 217%. The median lifespan was extended from 43 days to 78 days (range: 74-88 days) in the AAV1-GALC-treated group. Widespread expression of GALC protein and alleviation of KD neuropathology were detected in the CNS of the treated mice when examined at the moribund stage. Functionally, elevated levels of psychosine were completely normalized in the forebrain region of the treated mice. In the posterior region, which includes the mid- and the hindbrain, psychosine was reduced by an average of 77% (range: 53-93%) compared to the controls. Notably, psychosine levels in this region were inversely correlated with body weight and lifespan of AAV1-GALC-treated mice, suggesting that the degree of viral transduction of posterior brain regions following ventricular injection determined treatment efficacy on growth and survivability, respectively. Overall, our results suggest that viral vector delivery via the cerebroventricular system can partially correct psychosine accumulation in brain that leads to slower disease progression in KD.

Galactosylceramidase deficiency and pathological abnormalities in cerebral white matter of Krabbe disease.

Krabbe Disease (KD) is an autosomal recessive disorder that results from loss-of-function mutations in the GALC gene, which encodes lysosomal enzyme galactosylceramidase (GALC). Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades. Despite the central role of GALC in KD pathomechanism, investigations of GALC deficiency at a protein level are largely absent, due in part, to the lack of sensitive antibodies in the field. Leveraging two custom antibodies that can detect GALC at endogenous levels, we demonstrated that GALC protein is predominantly localized to oligodendrocytes in cerebral white matter of an infant brain, consistent with its functional role in myelination. Mature GALC could also be quantitatively detected as a 26 kDa band by western blotting and correlated to enzyme activity in brain tissues. The p.Ile562Thr polymorphic variant, which is over-represented in the KD population, was associated with reduced mature GALC protein and activity. In three infantile KD cases, homozygous null mutations in GALC lead to deficiency in total GALC protein and activity. Interestingly, although GALC activity was absent, normal levels of total GALC protein were detected by a sandwich ELISA using our custom antibodies in a later-onset KD brain, which suggests that the assay has the potential to differentiate infantile- and later-onset KD cases. Among the infantile KD cases, we quantified a 5-fold increase in psychosine levels, and observed increased levels of acid ceramidase, a key enzyme for psychosine production, and hyperglycosylated lysosomal-associated membrane protein 1, a marker for lysosomal activation, in periventricular white matter, a major pathological brain region, when compared with age-matched normal controls. While near complete demyelination was observed in these cases, we quantified that an early-infantile case (age of death at 10 months) had about 3-fold increases in both globoid cells, a pathological hallmark for KD, and CD8-positive T lymphocytes, a pathological marker for multiple sclerosis, in the white matter when compared with a slower progressing infantile case (age of death at 21 months), which suggests a positive correlation between clinical severity and neuropathology. Taken together, our findings have advanced the understanding of GALC protein biology in the context of normal and KD brain white matter. We also revealed new neuropathological changes that may provide insights to understand KD pathogenesis.

Specificity and Diagnostic Utility of Cerebrospinal Fluid CXCL13 in Lyme Neuroborreliosis.

BACKGROUND: Demonstration of intrathecal production of Borrelia-specific antibodies (ITAb) is considered the most specific diagnostic marker of Lyme neuroborreliosis (LNB). Limitations include delayed detectability in early infection and continued presence long after successful treatment. Markers of active inflammation-increased cerebrospinal fluid (CSF) leukocytes, protein, and CXCL13-provide nonspecific markers of active infection. To assess the utility of CSF CXCL13, we measured its concentration in 132 patients with a broad spectrum of neuroinflammatory disorders, including LNB. METHODS: CSF CXCL13 was measured by immunoassay. Spearman rank correlation test was performed to explore its relationship to conventional markers of neuroinflammation and Borrelia-specific ITAb production. RESULTS: In non-LNB neuroinflammatory disorders, CSF CXCL13 elevation correlated with CSF immunoglobulin G (IgG) synthesis and leukocyte count. In LNB, CXCL13 concentration was far greater than expected from overall CSF IgG synthesis, and correlated with Borrelia-specific ITAb synthesis. Median CSF CXCL13 concentration in ITAb-positive LNB patients was > 500 times greater than in any other group. CONCLUSIONS: Intrathecal CXCL13 and IgG production are closely interrelated. CXCL13 is disproportionately increased in "definite LNB," defined as having demonstrable Borrelia-specific ITAb, but not "probable LNB," without ITAb. This disproportionate increase may help identify patients with very early infection or those with active vs treated LNB, or may help to differentiate ITAb-defined active LNB from other neuroinflammatory disorders. However, its reported specificity is closely related to the diagnostic requirement for ITAb. It may add little specificity to the demonstration of a pleocytosis or increased overall or specific IgG production in the CSF.

Neuroimmunomodulators in Neuroborreliosis and Lyme Encephalopathy.

Background: Lyme encephalopathy, characterized by nonspecific neurobehavioral symptoms including mild cognitive difficulties, may occur in patients with systemic Lyme disease and is often mistakenly attributed to central nervous system (CNS) infection. Identical symptoms occur in many inflammatory states, possibly reflecting the effect of systemic immune mediators on the CNS. Methods: Multiplex immunoassays were used to measure serum and cerebrospinal fluid (CSF) cytokines in patients with or without Lyme disease to determine if there are specific markers of active CNS infection (neuroborreliosis), or systemic inflammatory mediators associated with neurobehavioral syndromes. Results: CSF CXCL13 levels were elevated dramatically in confirmed neuroborreliosis (n = 8), less so in possible neuroborreliosis (n = 11) and other neuroinflammatory conditions (n = 44). Patients with Lyme (n = 63) or non-Lyme (n = 8) encephalopathy had normal CSF findings, but had elevated serum levels of interleukins 7, 17A, and 17F, thymic stromal lymphopoietin and macrophage inflammatory protein-α. Conclusions: CSF CXCL13 is a sensitive and specific marker of neuroborreliosis in individuals with Borrelia-specific intrathecal antibody production. However, it does not distinguish individuals strongly suspected of having neuroborreliosis, but lacking confirmatory intrathecal antibodies, from those with other neuroinflammatory conditions. Patients with mild cognitive symptoms occurring during acute Lyme disease, and/or after appropriate treatment, have normal CSF but elevated serum levels of T-helper 17 markers and T-cell growth factors, which are also elevated in patients without Lyme disease but with similar symptoms. In the absence of CSF abnormalities, neurobehavioral symptoms appear to be associated with systemic inflammation, not CNS infection or inflammation, and are not specific to Lyme disease.

Hepatic arterial reconstruction for orthotopic liver transplantation in the rat.

BACKGROUND: Orthotopic liver transplantation (OLT) models in rats have been investigated in many studies. The reconstruction of hepatic artery is required for reliable OLT and also requires advanced skills. METHODS: The hepatic artery reconstructions by a hand-suture technique and a new method using a micro T-tube were investigated in rats with a whole-liver syngeneic graft. Operative time and postoperative patency were compared between the hand-suture and micro T-tube techniques. RESULTS: Our technique using the micro T-tube shortened the operative time of recipient surgery compared with the hand-suture technique and prolonged the operative time for the donor. The patency ratio was maintained at 24h after OLT with hand suturing but was significantly reduced with the micro T-tube, which had a patency ratio of 0.83 only up to 6h after OLT. CONCLUSION: The micro T-tube technique may have potential usefulness in the rat OLT model but requires further modification.

Comprehensive and innovative techniques for liver transplantation in rats: a surgical guide.

AIM: To investigate our learning curves of orthotopic liver transplantation (OLT) in rats and the most important factor for successful surgery. METHODS: We describe the surgical procedures for our rat OLT model, and determined the operator learning curves. The various factors that contributed to successful surgery were determined. The most important surgical factors were evaluated between successful and unsuccessful surgeries. RESULTS: Learning curve data indicated that 50 cases were required for operator training to start a study. Operative time, blood loss, warm ischemic time, anhepatic phase, unstable systemic hemodynamic state, and body temperature after surgery significantly affected surgery success by univariate analysis, while the anhepatic phase was the most critical factor for success by multivariate analysis. CONCLUSION: OLT in rats is the only liver transplantation model that provides clinically relevant and reliable results. Shortened anhepatic phase is key to success in this model.

Molecular characterization of mutations that cause globoid cell leukodystrophy and pharmacological rescue using small molecule chemical chaperones.

Globoid cell leukodystrophy (GLD) (Krabbe disease) is an autosomal recessive, degenerative, lysosomal storage disease caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. Of the >70 disease-causing mutations in the GALC gene, most are located outside of the catalytic domain of the enzyme. To determine how GALC mutations impair enzymatic activity, we investigated the impact of multiple disease-causing mutations on GALC processing, localization, and enzymatic activity. Studies in mammalian cells revealed dramatic decreases in GALC activity and a lack of appropriate protein processing into an N-terminal GALC fragment for each of the mutants examined. Consistent with this, we observed significantly less GALC localized to the lysosome and impairment in either the secretion or reuptake of mutant GALC. Notably, the D528N mutation was found to induce hyperglycosylation and protein misfolding. Reversal of these conditions resulted in an increase in proper processing and GALC activity, suggesting that glycosylation may play a critical role in the disease process in patients with this mutation. Recent studies have shown that enzyme inhibitors can sometimes "chaperone" misfolded polypeptides to their appropriate target organelle, bypassing the normal cellular quality control machinery and resulting in enhanced activity. To determine whether this may also work for GLD, we examined the effect of alpha-lobeline, an inhibitor of GALC, on D528N mutant cells. After treatment, GALC activity was significantly increased. This study suggests that mutations in GALC can cause GLD by impairing protein processing and/or folding and that pharmacological chaperones may be potential therapeutic agents for patients carrying certain mutations.

Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts.

This paper describes the bioconjugation of histidine-tagged enzymes and other proteins to the surface of composite "magnetomicelles" consisting of magnetic gamma-Fe2O3 nanoparticles encapsulated within cross-linked polystyrene-block-polyacrylate copolymer micelle shells. Free carboxylic acid groups on the magnetomicelle surface were converted to Cu2+-iminodiacetic acid (IDA) for protein capture. The conjugation of T4 DNA ligase and enhanced green fluorescent protein to magnetomicelles revealed that proteins were captured with a high surface density and could be magnetically separated from reaction mixtures and subsequently released from the nanoparticle surface. Additionally, bioconjugation of T7 RNA polymerase yielded a functional enzyme that maintained its biological activity and could be recycled for up to three subsequent transcription reactions. We propose that protein-magnetomicelle bioconjugates are effective for protein bioseparation and enzymatic recycling and further strengthen the idea that nanoparticle surfaces have utility in protein immobilization.

DNA dissociation and degradation at gold nanoparticle surfaces.

The instability of DNA-functionalized gold nanoparticles (Au-DNA conjugates) upon exposure to high temperatures is characterized using fluorescence spectroscopy, gel electrophoresis and ion-exchange chromatography. Above 70 degrees C, aqueous Au-DNA conjugates decompose within hours due to both desorption of thiol-terminated DNA from the gold nanoparticle surface and chemical degradation of DNA in the presence of colloidal gold. Although the chemical mechanism for DNA degradation was not identified in this study, the gold surface participates directly in the cleavage reaction. These results have important implications for the use of Au-DNA conjugates in biotechnological and clinical applications that require high temperatures, such as polymerase chain reaction (PCR).