Functional Protein Delivery Using Polymeric Nanoparticles: A Novel Therapeutic Approach to Alpha-Synuclein Aggregation and Parkinson's Disease

203 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson's disease (PD). Synthetic nanoparticles made from polybutylcyanoacrylate (PBCA) have been previously shown to deliver otherwise impermeable drugs to therapeutic levels in vivo across the blood-brain barrier (BBB). It was not yet known, however, whether such particles could also be taken up by neurons and other brain cells. Here we report the novel use of polybutylcyanoacrylate nanoparticles (NPs) for delivery of intact, functional proteins into neurons and neurogenic cell lines. Uptake of these particles is primarily dependent on endocytosis via the low-density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant E. coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. In addition, a monoclonal antibody (H3C) directed against the C-terminus of synuclein is capable of interacting with endogenous alpha-synuclein (alpha-syn) in neurons following transport via polybutylcyanoacrylate NPs.Alpha-synuclein (alpha-syn) aggregation has been linked to the pathogenesis of PD and other related conditions. Targeting this protein using synuclein-specific antibodies has thus emerged as a promising strategy for treatment development, but is limited by the need for rigorous protein expression in neuronal cells. We therefore explore the effects of treatment with H3C-loaded NPs in a cell-based model of Parkinson's disease. H3C reduces alpha-syn aggregate levels and cytotoxicity during oxidative stress. This process is dependent on lysosomal activity and is abolished by lysosomal inhibition. Binding to H3C also increases degradation of mutant forms of alpha-synuclein by purified lysosomes in vitro. H3C-loaded nanoparticles may thus aid in our understanding of abnormal protein turnover in cells, and offer a novel therapeutic approach to alpha-syn misfolding diseases such as PD.Finally, we examine the regional distribution and cellular uptake of PBCA nanoparticles in vivo following intravenous administration in mice. The particles are most strongly localized to the cerebellum, midbrain, entorrhinal cortex, hippocampus, subventricular zone, and striatum. Cell-specific staining was also observed in Purkinje neurons of the cerebellum. Positive enzyme activity was furthermore detected in the olfactory bulb of animals injected with beta-galactosidase loaded NPs. Future studies are necessary, however, in order to achieve more concentrated and targeted delivery of cargo to brain regions specifically affected by Parkinson's disease, and for optimizing neuronal internalization of the nanoparticles following passage across the BBB. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro, and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo .U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Functional protein delivery into neurons using polymeric nanoparticles

An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo

Profound intellectual disability caused by homozygous TRAPPC9 pathogenic variant in a man from Malta

Abstract Background Intellectual disability is a complex multi‐faceted condition with diverse underlying etiologies. One rare form of intellectual disability is secondary to the loss of TRAPPC9, an activator of NF‐κB and a mediator of intracellular protein processing and trafficking. TRAPPC9 deficiency has been described in 48 patients with more than 15 pathologic variants. Method Clinical evaluation, magnetic resonance imaging, and whole‐exome sequencing were used to characterize the underlying cause of absent speech, restricted/repetitive behaviors, and worsening behavioral outbursts in 27‐year‐old man from Malta. Results Magnetic Resonance Imaging showed morphologic abnormalities, including global cerebral and cerebellar hypoplasia. Genetic analysis through Whole Exome Sequencing identified a homozygous deletion (c.568_574del) in TRAPPC9 resulting in a frameshift, premature stop codon, and ultimately a truncated protein (p.Trp190Argfs*95). In this case, the pathogenic variant was homozygous, identified in both of the parents without known consanguinity. Conclusion Given the phenotype and genotype consistent with a deficiency in TRAPPC9, it is likely that this patient represents a novel case of this rare genetic syndrome. Specifically, this case, in the context of 48 total reported patients, raises questions as to the geographic origin of the pathologic variant and optimal detection and therapeutic intervention for this condition

Compound heterozygosity with a novel S222N GALT mutation leads to atypical galactosemia with loss of GALT activity in erythrocytes but little evidence of clinical disease

Galactosemia is an inborn error of galactose metabolism caused by mutations in the GALT gene. Though early detection and galactose restriction prevent severe liver disease, affected individuals have persistently elevated biomarkers and often neuro-developmental symptoms. We present a teenage compound heterozygote for a known pathogenic mutation (H132Q) and a novel variant of unknown significance (S222N), with nearly absent erythrocyte GALT enzyme activity but normal biomarkers and only mild anxiety despite diet non-adherence. This case is similar to a previously reported S135L mutation. In this report we investigate the novel S222N variant and critically evaluate a clinically puzzling case

Molecular characterization and reclassification of a 1.18 Mbp DMD duplication following positive carrier screening for Duchenne/Becker muscular dystrophy

Abstract A 2‐month‐old male patient harboring a duplication of DMD exons 1–7 classified as pathogenic by an outside institution presented with mildly elevated creatine phosphokinase (CK); molecular breakpoint analysis by our laboratory reclassified the duplication as likely benign. To date, proband continues to develop normally with decreased CK, further supporting our reclassification