Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11

Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work withZfyve26−/− mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction

The synthesis and study of an amine functionalized crown ether

This study has resulted in a route to the first known NHZ functionalized xylenebased crown ether, 5-amino-2-methoxy-1,3-xylyl-18-crown-5. The route involves preparing 5-azido-2-methoxy-1,3-xylyl-18-crown-5 from 5-bromo-2-methoxy-1,3-xylyl18-crown-5 by reacting it in turn with n-BuLi and tosyl azide. 5-Amino-2-methoxy-1,3xylyl-l8-crown-5 was obtained by reducing 5-azido-2-methoxy-1,3-xylyl-l8-crown-5 with aqueous sodium borohydride in the presence of a phase transfer agent. The 'H NMR spectrum of the amino derivative showed NMR signals at 6 3.4-3.7 (crown CHZ), S 4.0 (benzylic), S 4.47 (methoxy), and 6 6.58 (aromatic) ppm. The integrated areas were consistent with the formula, and they also suggested the NH2 protons were in the crown CH2 area. The IR (KBr pellet) spectrum showed bands at 3408 cm' and 3364 cm' corresponding to the N-H asymmetric and symmetric stretches, respectively. This study has also provided a new procedure for the preparation of 4-bromo-2,6-bis(bromomethyl) anisole, which was the intermediate for 5-bromo-2-methoxy-1,3-xylyl-18-crown-5. It involved reacting 4-bromophenol in turn with 30 % formaldehyde, dimethylsulfate, and HBr in acetic acid.Thesis (M.S.)Department of Chemistr

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the ?-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis\u2013Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay\u2013Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant ?-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.Peer reviewed: YesNRC publication: Ye

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels

Rapid Assembly of a Library of Lipophilic Iminosugars via the Thiol–Ene Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease

A highly divergent route to lipophilic iminosugars that utilizes the thiol–ene reaction was developed to enable the rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing various different lipophilic substituents. Enzyme kinetic analyses revealed that a number of these products are potent, low-nanomolar inhibitors of human glucocerebrosidase that stabilize the enzyme to thermal denaturation by up to 20 K. Cell based assays conducted on Gaucher disease patient derived fibroblasts demonstrated that administration of the compounds can increase lysosomal glucocerebrosidase activity levels by therapeutically relevant amounts, as much as 3.2-fold in cells homozygous for the p.N370S mutation and 1.4-fold in cells homozygous for the p.L444P mutation. Several compounds elicited this increase in enzyme activity over a relatively wide dosage range. The data assembled here illustrate how the lipophilic moiety common to many glucocerebrosidase inhibitors might be used to optimize a lead compound’s ability to chaperone the protein in cellulo. The flexibility of this synthetic strategy makes it an attractive approach to the rapid optimization of glycosidase inhibitor potency and pharmacokinetic behavior

Rapid Assembly of a Library of Lipophilic Iminosugars via the Thiol–Ene Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease

A highly divergent route to lipophilic iminosugars that utilizes the thiol–ene reaction was developed to enable the rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing various different lipophilic substituents. Enzyme kinetic analyses revealed that a number of these products are potent, low-nanomolar inhibitors of human glucocerebrosidase that stabilize the enzyme to thermal denaturation by up to 20 K. Cell based assays conducted on Gaucher disease patient derived fibroblasts demonstrated that administration of the compounds can increase lysosomal glucocerebrosidase activity levels by therapeutically relevant amounts, as much as 3.2-fold in cells homozygous for the p.N370S mutation and 1.4-fold in cells homozygous for the p.L444P mutation. Several compounds elicited this increase in enzyme activity over a relatively wide dosage range. The data assembled here illustrate how the lipophilic moiety common to many glucocerebrosidase inhibitors might be used to optimize a lead compound’s ability to chaperone the protein in cellulo. The flexibility of this synthetic strategy makes it an attractive approach to the rapid optimization of glycosidase inhibitor potency and pharmacokinetic behavior

Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11.

ObjectiveHereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism.MethodsWe describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes.ResultsFibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent.InterpretationEmerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26-/- mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction

Amino acid changes mage in the H1 and H2 alpha-beta hybrid subunits.

1<p>Aligned amino acids from the alpha subunit that were substituted to make the hybrid.</p>2<p>Also enhances MUGS binding and hydrolysis.</p

DEAE ion-exchange separation of the Hex isozymes from the lysate of transfected feline SD fibroblasts highly expressing the H1 beta-alpha hybrid (similar results were obtained using lysate from these cells expressing the H2 hybrid, thus a generic “H” is used for the hybrid subunit).

<p>DEAE ion-exchange separation of the Hex isozymes from the lysate of transfected feline SD fibroblasts highly expressing the H1 beta-alpha hybrid (similar results were obtained using lysate from these cells expressing the H2 hybrid, thus a generic “H” is used for the hybrid subunit).</p