17 research outputs found
Protein Synthesis-Dependent Associative Long-Term Memory in Larval Zebrafish
The larval zebrafish is a model organism to study the neural circuitry underlying behavior. There exist, however, few examples of robust long-term memory. Here we describe a simple, unrestrained associative place-conditioning paradigm. We show that visual access to a group of conspecifics has rewarding properties for 6- to 8-day-old larval zebrafish. We use this social reward as an unconditioned stimulus and pair it with a distinct visual environment. After training, larvae exhibited spatial preference for the location previously paired with the social reward for up to 36 h, indicating that zebrafish larvae can exhibit long-term associative memory. Furthermore, incubation with a protein synthesis inhibitor or an NMDAR-antagonist impaired memory. In future experiments, this learning paradigm could be used to study the social interactions of larval zebrafish or paired with cell-specific metabolic labeling to visualize circuits underlying memory formation
Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish
Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells’ own translational machinery. Using the selective “click reaction”, this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that noncanonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae
Molecular Genetics of Neurodegenerative Dementias.
Neurodegenerative dementias are clinically heterogeneous, progressive diseases with frequently overlapping symptoms, such as cognitive impairments and behavior and movement deficits. Although a majority of cases appear to be sporadic, there is a large genetic component that has yet to be fully explained. Here, we review the recent genetic and genomic findings pertaining to Alzheimer's disease, frontotemporal dementia, Lewy body dementia, and prion dementia. In this review, we describe causal and susceptibility genes identified for these dementias and discuss recent research pertaining to the molecular function of these genes. Of particular interest, there is a large overlap in clinical phenotypes, genes, and/or aggregating protein products involved in these diseases, as well as frequent comorbid presentation, indicating that these dementias may represent a continuum of syndromes rather than individual diseases
Recommended from our members
Molecular Genetics of Neurodegenerative Dementias.
Neurodegenerative dementias are clinically heterogeneous, progressive diseases with frequently overlapping symptoms, such as cognitive impairments and behavior and movement deficits. Although a majority of cases appear to be sporadic, there is a large genetic component that has yet to be fully explained. Here, we review the recent genetic and genomic findings pertaining to Alzheimer's disease, frontotemporal dementia, Lewy body dementia, and prion dementia. In this review, we describe causal and susceptibility genes identified for these dementias and discuss recent research pertaining to the molecular function of these genes. Of particular interest, there is a large overlap in clinical phenotypes, genes, and/or aggregating protein products involved in these diseases, as well as frequent comorbid presentation, indicating that these dementias may represent a continuum of syndromes rather than individual diseases
Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish
Protein expression in the nervous system undergoes regulated
changes
in response to changes in behavioral states, in particular long-term
memory formation. Recently, methods have been developed (BONCAT and
FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal
functional groups into proteins using the cells’ own translational
machinery. Using the selective “click reaction”, this
allows for the identification and visualization of newly synthesized
proteins in vitro. Here we demonstrate that noncanonical amino acid
labeling can be achieved in
vivo in an intact organism capable of simple learning behavior, the
larval zebrafish. We show that azidohomoalanine is metabolically incorporated
into newly synthesized proteins, in a time- and concentration-dependent
manner, but has no apparent toxic effect and does not influence simple
behaviors such as spontaneous swimming and escape responses. This
enables fluorescent labeling of newly synthesized proteins in whole
mount larval zebrafish. Furthermore, stimulation with a GABA antagonist
that elicits seizures in the larval zebrafish causes an increase in
protein synthesis throughout the proteome, which can also be visualized
in intact larvae
Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish
Protein expression in the nervous system undergoes regulated
changes
in response to changes in behavioral states, in particular long-term
memory formation. Recently, methods have been developed (BONCAT and
FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal
functional groups into proteins using the cells’ own translational
machinery. Using the selective “click reaction”, this
allows for the identification and visualization of newly synthesized
proteins in vitro. Here we demonstrate that noncanonical amino acid
labeling can be achieved in
vivo in an intact organism capable of simple learning behavior, the
larval zebrafish. We show that azidohomoalanine is metabolically incorporated
into newly synthesized proteins, in a time- and concentration-dependent
manner, but has no apparent toxic effect and does not influence simple
behaviors such as spontaneous swimming and escape responses. This
enables fluorescent labeling of newly synthesized proteins in whole
mount larval zebrafish. Furthermore, stimulation with a GABA antagonist
that elicits seizures in the larval zebrafish causes an increase in
protein synthesis throughout the proteome, which can also be visualized
in intact larvae
Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing.
Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. As one of the constituent lipids on LNP surfaces, PEGylated lipids (PEG-lipids) play an important role in defining LNP physicochemical properties and biological interactions. Previous studies indicate that LNP performance is modulated by tuning PEG-lipid parameters including PEG size and architecture, carbon tail type and length, as well as the PEG-lipid molar ratio in LNPs. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables. To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). A library of 54 ASO-LNP formulations with distinct PEG-lipid compositions was prepared using a liquid handling robot and assessed for their physiochemical properties as well as gene silencing efficacy in murine cortical neurons. Our results show that the molar ratio of anionic PEG-lipid in LNPs regulates particle size and PEG-lipid carbon tail length controls ASO-LNP gene silencing activity. ASO-LNPs formulated using PEG-lipids with optimal carbon tail lengths achieved up to 5-fold lower mRNA expression in neurons as compared to naked ASO. Representative ASO-LNP formulations were further characterized using dose-response curves and small-angle X-ray scattering to understand structure-activity relationships. Identified hits were also tested for efficacy in primary murine microglia and were scaled-up using a microfluidic formulation technique, demonstrating a smooth translation of ASO-LNP properties and in vitro efficacy. The reported HTS workflow can be used to screen additional multivariate parameters of LNPs with significant time and material savings, therefore guiding the selection and scale-up of optimal formulations for nucleic acid delivery to a variety of cellular targets
Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection
Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs
Identification of evolutionarily conserved gene networks mediating neurodegenerative dementia
Identifying the mechanisms through which genetic risk causes dementia is an imperative for new therapeutic development. Here, we apply a multistage, systems biology approach to elucidate the disease mechanisms in frontotemporal dementia. We identify two gene coexpression modules that are preserved in mice harboring mutations in MAPT, GRN and other dementia mutations on diverse genetic backgrounds. We bridge the species divide via integration with proteomic and transcriptomic data from the human brain to identify evolutionarily conserved, disease-relevant networks. We find that overexpression of miR-203, a hub of a putative regulatory microRNA (miRNA) module, recapitulates mRNA coexpression patterns associated with disease state and induces neuronal cell death, establishing this miRNA as a regulator of neurodegeneration. Using a database of drug-mediated gene expression changes, we identify small molecules that can normalize the disease-associated modules and validate this experimentally. Our results highlight the utility of an integrative, cross-species network approach to drug discovery