# US 8,785,149 B2 · In vitro cellular bioassay for neurotoxicity testing

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,785,149 B2
- Document ID: US8785149B2
- Status: Granted
- Kind: B2
- Application number: 13/639,541
- Filed: 2011-04-08
- Published: 2013-08-15
- Granted: 2014-07-22
- Earliest claimed: 2010-04-08
- Assignees: Arizona Board of Regents on Behalf of the University of Arizona
- Applicants: Linda L. Restifo; Robert Kraft
- Inventors: Linda L. Restifo; Robert Kraft
- Web page: https://patentgrep.com/patent/US8785149B2

## CPC

- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- G01N 33/5058: {Neurological cells}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides neurotoxicity and developmental neurotoxicity screening methods employing primary cultured neurons from Drosophila.

## Claims

### Claim 1

A method for determining the neurotoxicity of a compound comprising
a) culturing Drosophila neurons in the presence of said compound; and
b) assessing at least one characteristic of the neurons selected from the group of characteristics consisting of cell survival, neuronal body size, neuronal body shape, neurite outgrowth, neurite structure, neurite arbor shape, synapse number, and synapse function;
wherein a modulation of at least one characteristic of the neuron compared to Drosophila neurons cultured in the absence of the compound indicates that said compound is neurotoxic.

### Claim 2 (depends on 1)

The method of claim 1, wherein said Drosophila neurons are obtained from all or part of the central nervous system of Drosophila.

### Claim 3 (depends on 2)

The method of claim 2, wherein the Drosophila expresses at least one marker for the identification of a neuronal subtype.

### Claim 4 (depends on 3)

The method of claim 3, wherein said neuronal subtype is a mushroom body neuron.

### Claim 5 (depends on 1)

The method of claim 1, wherein said Drosophila neurons are obtained from Drosophila wandering larva or an earlier developmental stage.

### Claim 6 (depends on 1)

The method of claim 1, wherein step b) comprises observing said Drosophila neurons by optical microscopy.

### Claim 7 (depends on 1)

The method of claim 1, further comprising immunostaining said Drosophila neurons with antibodies immunologically specific for a neuron membrane antigen.

### Claim 8 (depends on 1)

The method of claim 1, wherein said Drosophila neurons are mutant Drosophila neurons comprising at least one mutation in at least one gene.

### Claim 9 (depends on 8)

The method of claim 8, wherein said gene is selected from the group consisting of singed, rho-type guanine exchange factor (rtGEF), GDP dissociation inhibitor, Graf, p21-activated protein kinase (Pak), cheerio, mew, dystrophin, ubiquitin protein ligase E3A (Ube3a), nejire, tuberous sclerosis complex 1 (Tsc1), gigas, neurexin, neuroligin, Neurexin IV, and discs large (dlg).

### Claim 10 (depends on 8)

The method of claim 8, wherein said mutation causes an intellectual disability.

### Claim 11 (depends on 1)

The method of claim 1, wherein said compound is a HMG CoA reductase inhibitor.

### Claim 12 (depends on 11)

The method of claim 11, wherein step b) comprises assessing the neurons for a beads-on-a-string phenotype, wherein said beads-on-a-string phenotype comprises ovoid nodules spaced along the length of a neurite.

### Claim 13 (depends on 1)

The method of claim 1, wherein step b) comprises quantitating characteristics of the neurons with imaging software.

### Claim 14 (depends on 1)

The method of claim 1, wherein a reduction in neurite outgrowth compared to Drosophila neurons cultured in the absence of the compound indicates that said compound is neurotoxic.

### Claim 15 (depends on 1)

The method of claim 1, wherein step b) comprises assessing the neurons for a beads-on-a-string phenotype, wherein said beads-on-a-string phenotype comprises ovoid nodules spaced along the length of a neurite.

### Claim 16 (depends on 1)

The method of claim 1, wherein step a) comprises culturing Drosophila neurons in the presence of more than one concentration of said compound.

### Claim 17 (depends on 16)

The method of claim 16, wherein step a) comprises culturing Drosophila neurons in the presence of a concentration curve of said compound.