ABSTRACT
Canavan disease, a leukodystrophy caused by loss-of-function ASPA mutations, is characterized by brain dysmyelination, vacuolation, and astrogliosis ("spongiform leukodystrophy"). ASPA encodes aspartoacylase, an oligodendroglial enzyme that cleaves the abundant brain amino acid N-acetyl-L-aspartate (NAA) to L-aspartate and acetate. Aspartoacylase deficiency results in a 50% or greater elevation in brain NAA concentration ([NAAB]). Prior studies showed that homozygous constitutive knockout of Nat8l, the gene encoding the neuronal NAA synthesizing enzyme N-acetyltransferase 8-like, prevents aspartoacylase-deficient mice from developing spongiform leukodystrophy. We now report that brain Nat8l knockdown elicited by intracerebroventricular/intracisternal administration of an adeno-associated viral vector carrying a short hairpin Nat8l inhibitory RNA to neonatal aspartoacylase-deficient AspaNur7/Nur7 mice lowers [NAAB] and suppresses development of spongiform leukodystrophy.
Subject(s)
Acetyltransferases/genetics , Amidohydrolases/deficiency , Canavan Disease/genetics , Canavan Disease/metabolism , Animals , Brain/metabolism , Brain/pathology , Canavan Disease/pathology , Canavan Disease/physiopathology , Dependovirus/genetics , Disease Models, Animal , Gene Expression , Gene Knockdown Techniques , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Mice , Mice, Knockout , Motor Activity , Neurons/metabolism , RNA, Messenger/genetics , Transduction, GeneticABSTRACT
Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in AspaNur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like. SIGNIFICANCE STATEMENT: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.
Subject(s)
Aspartic Acid/analogs & derivatives , Canavan Disease/metabolism , Disease Models, Animal , Neurons/metabolism , Animals , Aspartic Acid/biosynthesis , Aspartic Acid/deficiency , Aspartic Acid/genetics , Canavan Disease/genetics , Canavan Disease/pathology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/pathologyABSTRACT
The authors report on series of side-chain smectic liquid crystal elastomer (LCE) cell scaffolds based on star block-copolymers featuring 3-arm, 4-arm, and 6-arm central nodes. A particular focus of these studies is placed on the mechanical properties of these LCEs and their impact on cell response. The introduction of diverse central nodes allows to alter and custom-modify the mechanical properties of LCE scaffolds to values on the same order of magnitude of various tissues of interest. In addition, it is continued to vary the position of the LC pendant group. The central node and the position of cholesterol pendants in the backbone of ε-CL blocks (alpha and gamma series) affect the mechanical properties as well as cell proliferation and particularly cell alignment. Cell directionality tests are presented demonstrating that several LCE scaffolds show cell attachment, proliferation, narrow orientational dispersion of cells, and highly anisotropic cell growth on the as-synthesized LCE materials.
Subject(s)
Biocompatible Materials/chemistry , Elastomers/chemistry , Liquid Crystals/chemistry , Mechanical Phenomena , Animals , Biocompatible Materials/chemical synthesis , Biocompatible Materials/pharmacology , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Dermis/cytology , Elastomers/chemical synthesis , Elastomers/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Liquid Crystals/ultrastructure , Mice , Microscopy, Polarization , Myoblasts/cytology , Myoblasts/drug effects , Porosity , Scattering, Small Angle , Stress, Mechanical , Temperature , X-Ray DiffractionABSTRACT
We report that liquid crystal elastomers (LCEs), often portrayed as artificial muscles, serve as scaffolds for skeletal muscle cell. A simultaneous microemulsion photopolymerization and cross-linking results in nematic LCE microspheres 10-30 µm in diameter that when conjoined form a LCE construct that serves as the first proof-of-concept for responsive LCE muscle cell scaffolds. Confocal microscopy experiments clearly established that LCEs with a globular, porous morphology permit both attachment and proliferation of C2C12 myoblasts, while the nonporous elastomer morphology, prepared in the absence of a microemulsion, does not. In addition, cytotoxicity and proliferation assays confirm that the liquid crystal elastomer materials are biocompatible promoting cellular proliferation without any inherent cytotoxicity.
Subject(s)
Biocompatible Materials/chemistry , Cell Adhesion/drug effects , Elastomers/chemistry , Liquid Crystals/chemistry , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/pharmacology , Cell Line , Cell Proliferation/drug effects , Elastomers/pharmacology , Mice , Microspheres , MyoblastsABSTRACT
Canavan disease is caused by inactivating ASPA (aspartoacylase) mutations that prevent cleavage of N-acetyl-L-aspartate (NAA), resulting in marked elevations in central nervous system (CNS) NAA and progressively worsening leukodystrophy. We now report that ablating NAA synthesis by constitutive genetic disruption of Nat8l (N-acetyltransferase-8 like) permits normal CNS myelination and prevents leukodystrophy in a murine Canavan disease model.
Subject(s)
Aspartic Acid/analogs & derivatives , Canavan Disease/metabolism , Canavan Disease/prevention & control , Disease Models, Animal , Animals , Aspartic Acid/deficiency , Aspartic Acid/genetics , Aspartic Acid/metabolism , Canavan Disease/genetics , Female , Male , Mice , Mice, KnockoutABSTRACT
Here we report on the modular synthesis and characterization of biodegradable, controlled porous, liquid crystal elastomers (LCE) and their use as three-dimensional cell culture scaffolds. The elastomers were prepared by cross-linking of star block-co-polymers with pendant cholesterol units resulting in the formation of smectic-A LCEs as determined by polarized optical microscopy, DSC, and X-ray diffraction. Scanning electron microscopy revealed the porosity of the as-prepared biocompatible LCEs, making them suitable as 3D cell culture scaffolds. Biodegradability studies in physiological buffers at varying pH show that these scaffolds are intact for about 11 weeks after which degradation sets in at an exponential rate. Initial results from cell culture studies indicate that these smectic LCEs are compatible with growth, survival, and expansion of cultured neuroblastomas and myoblasts when grown on the LCEs for extended time periods (about a month). These preliminary cell studies focused on characterizing the elastomer-based scaffolds' biocompatibility and the successful 3D incorporation as well as growth of cells in 60 to 150-µm thick elastomer sheets.