In vivo selection in non-human primates identifies superior AAV capsids for on-target CSF delivery to spinal cord.

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal delivery of AAV vectors into the cerebral spinal fluid (CSF) can avoid many of the issues of systemic delivery, although achieving broad distribution of the vector and transgene expression throughout the spinal cord is challenging and vector entry to the periphery occurs, sometimes initiating hepatotoxicity. Here we performed two rounds of in vivo biopanning in non-human primates (NHPs) with an AAV9 peptide display library injected intrathecally and performed insert sequencing on DNA isolated from either whole tissue (conventional selection), isolated nuclei, or nuclei from transgene-expressing cells. A subsequent barcoded pool of candidates and AAV9 was compared at the DNA (biodistribution) and RNA (expression) level in spinal cord and liver of intrathecally injected NHPs. Most of the candidates displayed enhanced biodistribution compared to AAV9 at all levels of spinal cord ranging from 2 to 265-fold. Nuclear isolation or expression-based selection yielded 4 of 7 candidate capsids with enhanced transgene expression in spinal cord (up to 2.4-fold), while no capsid obtained by conventional selection achieved that level. Furthermore, several capsids displayed lower biodistribution to the liver of up to 1,250-fold, compared to AAV9, providing a remarkable on target/off target biodistribution ratio. These capsids may have potential for gene therapy programs directed at the spinal cord and the selection method described here should be useful in clinically relevant large animal models.

The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.

Heterozygous mutations in the GBA1 gene - encoding lysosomal glucocerebrosidase (GCase) - are the most common genetic risk factors for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model used constitutive overexpression of wild-type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. Although increased aSyn pathology and grip strength reductions were observed in this model, the nigrostriatal system remained largely intact. The second model involved injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V knock-in mouse model. The GBA1 D409V mutation did not exacerbate the pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.

Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the GBA1 gene that encodes the lysosomal enzyme acid ß-glucosidase (GCase). Reduced GCase activity primarily leads to the accumulation of two substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Current treatment options have not been shown to ameliorate the neurological pathology observed in the most severe forms of GD, clearly representing an unmet medical need. To better understand the relationship between GlcCer and GlcSph accumulation and ultimately their connection with the progression of neurological pathology, we developed LC-MS/MS methods to quantify GlcCer and GlcSph in mouse brain tissue. A significant challenge in developing these methods was the chromatographic separation of GlcCer and GlcSph from the far more abundant isobaric galactosyl epimers naturally occurring in white matter. After validation of both methods, we evaluated the levels of both substrates in five different GD mouse models, and found significant elevation of brain GlcSph in all five, while GlcCer was elevated in only one of the five models. In addition, we measured GlcCer and GlcSph levels in the brains of wild-type mice after administration of the GCase inhibitor conduritol ß-epoxide (CBE), as well as the nonlysosomal ß-glucosidase (GBA2) inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ). Inhibition of GCase by CBE resulted in elevation of both sphingolipids; however, inhibition of GBA2 by NB-DGJ resulted in elevation of GlcCer only. Taken together, these data support the idea that GlcSph is a more selective and sensitive biomarker than GlcCer for neuronopathic GD in preclinical models.

A GCase chaperone improves motor function in a mouse model of synucleinopathy.

Mutation of the lysosomal hydrolase acid-ß-glucosidase (GCase), which leads to reduced GCase activity, is one of the most frequent genetic risk factors for Parkinson's disease (PD) and promotes α-synuclein accumulation in the brain, a hallmark of PD and other synucleinopathies. Whether targeting GCase pharmacologically is a valid therapeutic strategy for sporadic PD in the absence of GCase mutation is unknown. We have investigated whether increasing the stability, trafficking, and activity of wild-type GCase could be beneficial in synucleinopathies by administering the pharmacological chaperone AT2101 (afegostat-tartrate, isofagomine) to mice that overexpress human wild-type α-synuclein (Thy1-aSyn mice). AT2101 administered orally for 4 months to Thy1-aSyn mice improved motor and nonmotor function, abolished microglial inflammatory response in the substantia nigra, reduced α-synuclein immunoreactivity in nigral dopaminergic neurons, and reduced the number of small α-synuclein aggregates, while increasing the number of large α-synuclein aggregates. These data support the further investigation of pharmacological chaperones that target GCase as a therapeutic approach for sporadic PD and other synucleinopathies, even in the absence of glucocerebrosidase mutations.

Arp10p is a pointed-end-associated component of yeast dynactin.

In metazoans, dynein-dependent vesicle transport is mediated by dynactin, containing an actin-related protein, Arp1p, together with a cargo-selection complex containing a second actin-related protein, Arp11. Paradoxically, in budding yeast, models of dynactin function imply an interaction with membranes, whereas the lack of microtubule-based vesicle transport implies the absence of a cargo-selection complex. Using both genetic and biochemical approaches, we demonstrate that Arp10p is the functional yeast homologue of Arp11, suggesting the possible existence of a pointed-end complex in yeast. Specifically, Arp10p interacts with Arp1p and other dynactin subunits and is dependent on Arp1p for stability. Conversely, Arp10p stabilizes the dynactin complex by association with the Arp1p filament pointed end. Using a novel hRAS-Arp1p one-hybrid assay, we show that Arp1p associates with the plasma membrane dependent on dynactin subunits, but independent of dynein, and sensitive to cell wall damage. We directly show the association of Arp1p with not only the plasma membrane but also with a less dense membrane fraction. Based on the hRAS-Arp1p assay, loss of Arp10p enhances the apparent association of dynactin with the plasma membrane and suppresses the loss of signaling conferred by cell wall damage.

Alanine scanning of Arp1 delineates a putative binding site for Jnm1/dynamitin and Nip100/p150Glued.

Arp1p is the only actin-related protein (ARP) known to form actin-like filaments. Unlike actin, Arp1p functions with microtubules, as part of the dynein regulator, dynactin. Arp1p's dissimilar functions imply interactions with a distinct set of proteins. To distinguish surface features relating to Arp1p's core functions and to identify the footprint of protein interactions essential for dynactin function, we performed the first complete charge-cluster-to-alanine scanning mutagenesis of an ARP and compared the results with a similar study of actin. The Arp1p mutations revealed three nonoverlapping surfaces with distinct genetic properties. One of these surfaces encompassed a region unique to Arp1p that is crucial for Jnm1p (dynamitin/p50) and Nip100p (p150(Glued)) association as well as pointed-end associations. Unlike the actin mutations, none of the ARP1 alleles disrupt filament formation; however, one pointed-end allele delayed the elution of Arp1p on gel filtration, consistent with loss of additional subunits.

Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm.

Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryl-disulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm.