FIELD OF THE INVENTION The present invention relates to cultures of human olfactory neurons and to methods of screening agents for therapeutic and toxic effects using such cultures.

BACKGROUND OF THE INVENTION Neurons have eluded the attempts of researchers to maintain them in continuous cultures largely because most neurons do not divide and/or proliferate. Investigators have been able to maintain fetal or newborn rat neurons in culture, however, the neurons are static, do not divide and die after several months .

Olfactory sensory neurons, which are of central origin and are considered part of the central nervous system, do divide [Talamo et al., Nature 337:736-739, (1989)]. In fact, olfactory neurons from rats have been cultured [Coon et al., PNAS USA 86:1703-1707, (1989)]. However, rats do not develop human neurologic diseases, such as Alzheimer's disease and other human mental ill- nesses. Therefore, cultures of rat neurons can not be used to test for potential therapeutic agents for these human diseases. Furthermore, rats have different toler¬ ance levels of toxic compounds than humans . Therefore, the neurotoxicity of various agents to humans could be more accurately determined using cultured human neurons than cultured rat neurons.

Cell cultures of cancer cells, such as human neuroblastoma cells, also exist. However, these cells tend to be undifferentiated and do not exhibit a strong neuronal phenotype. Neuroblastomas are not of central nervous system origin. Furthermore, neuroblastomas have not been cultured from individuals with generalized central nervous system diseases (i.e., neuro/psychiatric illnesses ) . Human olfactory neurons, being primarily, of central nervous system origin from healthy individuals and individual with neuro/psychiatric illnesses would offer better systems for testing agents for therapeutic and

toxic effects.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide human olfactory neuron cultures from individuals without central nervous system diseases and from individuals with diseases of the central nervous system.

It is another object of the present invention to provide a method for assessing the potential neurotox- icity and potential therapeutic effects of agents in humans without employing live animals.

It is a further object of the present invention to provide a method for studying generalized diseases of the central nervous system. Various other object and advantages of the present invention will be apparent from the drawings and the following description of the invention.

In one embodiment, the present invention relates to a culture of replicating mammalian olfactory neurons, such as human. The neurons may display normal neuronal pathology or they may display pathology characteristic of a central nervous system disease.

In another embodiment, the present invention relates to a method of replicating mammalian olfactory neurons comprising contacting mammalian neurons with a growth medium and maintaining the neurons and medium under conditions allowing replication.

In a further embodiment, the present invention relates to a method of screening drugs for their ability to reverse or eliminate expression of central nervous system disease pathology comprising contacting cultured human neurons which show pathological characteristics of a central nervous system disease with a drug under condi¬ tions such that reversal or elimination of the disease pathology can be effected.

In yet another embodiment, the present invention relates to a method of screening drugs for their ability to inhibit virus activity on neurons comprising contacting

cultured human neurons with a drug under conditions such that inhibition of viral function can be effected.

In yet a further embodiment, the present invention relates to a method of testing for neurotoxicity of an agent comprising contacting replicating human olfactory neurons with an agent and determining the toxic effect of the agent on the neurons.

In another embodiment, the present invention relates to a method of diagnosing Alzheimer's disease. Human tissue containing olfactory neurons is isolated and the tissue is grown in a suitable medium under a first membrane comprising collagen or laminin. The neurons are then separated from the first membrane and replaced on a surface coated with a second membrane comprising collagen or laminin. The neurons are then cultured under condi¬ tions allowing replication. The culturing neurons are contacted with a calcium salt and then with a calcium ionophore. The presence or absence of Alzheimer's spe¬ cific protein are then detected. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the morphology of neurons grown in culture under conditions allowing for growth in three dimensions.

Figure IB shows the same neurons when grown on a basement membrane coated flat surface.

Figure 1C shows epithelial cells grown from the tissue sample on a basement membrane coated flat surface.

Figure ID and Figure IE show other cells not fully characterized grown on a basement membrane coated flat surface.

Figure 2 shows Western Blots of neuron specific proteins.

Figure 3 shows an immunoblot of calcium induced amyloid precursor protein. Note the doublet for 4250 + Ca lanes and the widened band for 90-106 + Ca lanes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to cultures of human olf ctory neurons and uses thereof. In one embodiment of

the present invention, cultures of human olfac-tory neurons are established from human tissue samples contain¬ ing neurons, such as nasal epithelium. Suitable tissue samples are obtained from the cadavers of healthy individ- uals that is to say individuals without any central nervous system diseases. Cultured neurons thus obtained, display normal neuronal pathology and express several neuron specific proteins (e.g., neuron specific enolase, neurofilament (68 D form) and tau protein). In another embodiment of the present invention, cultures of human olfactory neurons are established from a tissue sample obtained from the cadavers of individuals with Alzheimer's disease. It is anticipated that these cultures of neurons will display the pathology associated with Alzheimer's disease [Talamo et al., Nature 337:736- 739, (1989)]. As one skilled in the art will appreciate, cultures of neurons can be established using the method of the present invention from an individual afflicted with any generalized central nervous system disease which includes neurological and psychiatric illnesses such as Parkinson's disease, Tay-Sachs disease, schizophrenia, manic-depression, depression or mental retardation. A generalized central nervous system disease affects many types of neurons in the central nervous system including olfactory neurons. These cultured neurons may display the pathology of the disease affecting the individual from which the culture was grown.

In a preferred embodiment, the neuron cultures are established utilizing a basement membrane. The basement membrane consists mainly of laminin and collagen. There¬ fore, the present invention contemplates cultured human neurons which are initially grown and/or maintained on a membrane of laminin, collagen, related peptides thereof or a combination of the foregoing. When maintained under conditions allowing growth in three dimensions in the membrane the neurons appear more differentiated.

The neurons of the present invention are grown in Coon's 4506 medium. In addition to neurons, Coon's 4506

medium has been shown to support the growth of other human and rat cells. For example, using standard culture techniques and Coon's 4506 medium, human Pancreatic Islet cells, human cervical carcinoma cells, a colon cancer cell strain and rat cells have been grown. The present inven¬ tion also relates to cultures of transformed human olfac¬ tory neurons. One skilled in the art can easily transform and thereby immortalize cultured human olfactory neurons of the present invention without undue experimentation. Cultured neurons of the present invention can be trans¬ formed using, for example, the basic protocol described by Fredericksen et al. [Neuron 1:439-448, (1988)]. Neurons can be transformed using a retroviral vector, such as pZipNeoSVC(X) , which contains a temperature sensitive form of the SV40 large T antigen ts A58; the large T antigen is an oncogene. The transformed neurons will proliferate indefinitely in a relatively undifferentiated state at the permissive temperature of 33°C but will be able to differ¬ entiate into neurons at the nonpermissive temperature of 39°C.

In another embodiment of the present invention, cultured neurons from an individual with a central nervous system disease are used for screening drugs for their ability to reverse or eliminate the disease pathology. The cultured neurons are contacted with various drugs under conditions such that reversal or elimination of the disease pathology can be effected by the drug if it has the ability to do so.

For example, levels of dopamine receptors and/or receptor function in cells from a schizophrenic indivi¬ dual can be compared to that of normal individuals. If differences are found, then these differences can be used in assays to study the disease and to screen for drugs that correct the alteration. The presence of paired helical filaments or other Alzheimer's related antigens such as A68 can be used to assay for Alzheimer's disease pathology [Wolozin et al. , Science 232:648-650, (1986)]. In manic-depression alterations in G protein function are

hypothesized. Neurons of the present invention cultured from an individual with manic-depression can be used to screen for drugs correcting the G protein misfunction. Cultured neurons can also manifest the disorder present in gangliosidoses like Tay-Sachs disease, inherited forms of mental retardation or other inherited neurologic diseases [Adams et al., Principles of Neurology. (McGraw-Hill, New York. 1985). pp.718-759]. Neurons of the present inven¬ tion can be used to screen for drugs decreasing the build up of gangliosides .

The ability of drugs to reverse or eliminate Alzheimer's disease can be determined by their ability to reduce or eliminate the production of Alzheimer specific proteins, such as amyloid precursor proteins (APPs) and A68 protein. For testing potential therapeutically active compounds, cultured neurons from an individual with Alzheimer's disease are contacted with various drugs under conditions such that production of the specific proteins can be effected. Reduction or elimination of APPs or A68 is detected by preincubating the cultures in a calcium salt, such as CaCl ₂ , and then contacting the cultured neurons with a calcium ionophore, such as CA23187 or ionomycin, at a concentration sufficient to activate calcium dependent proteins. Calcium is a second messenger in the cells. The stimulation of other second messengers may also elicit Alzheimer's specific changes.

The presence or absence of APPs or A68 is detectedd by methods known in the art, for example, immunoblot techniques using an anti-A4 antibody or Alz-50 antibody. In addition, probes to the Alzheimer specific proteins can be developed using cloning, PCR or immuno- logic methods to facilitate detection.

Neurons of the present invention which naturally or through a modification secrete dopamine can be used for injection into the sybstantia nigra of individual with Parkinson's disease.

In another embodiment of the present invention, cultured neurons are used to screen for drugs that inhibit

viral activity in neurons. Neurons of the present inven¬ tion are contacted with a virus which naturally infects human neurons, such as the herpes virus or cytomegalovirus (CMV), and with various drugs under conditions such that inhibition of viral function can be effected. Drugs which inhibit virus attachment, penetration, replication or toxicity can be screened for and could be use in treatment or prevention of such infections.

Rat olfactory neurons can be stimulated to release the calcitonin gene related peptide (CGRP) by altering potassium levels when incubated with a neuro-toxin. Release of CGRP may be a sensitive indicator of neurotox- icity. Human olfactory neurons of the present invention containing CGRP or any other neurotransmitter can be used to assess the neurotoxicity of an agent by detecting alterations of basal and potassium evoked neurotransmitter release into the medium in the presence and absence of the agent. Neurotoxicity may correlate with a decline in neurotransmitter release over time as neuronal function deteriorates.

In another embodiment of the present invention, the cultured neurons are used for testing the neurotoxic¬ ity of an agent in humans. As one skilled in the art will appreciate, determining the effect of the agent on any of a number of neuronal characteristics will reflect that neurotoxicity of the agent. For example, the concentra¬ tion of the agent producing death in 50% of the neurons (LD50) can be determined using methods known in the art. The effect of the agent on neuronal growth characteristics such as population doubling time and length of processes also reflect the toxicity of the agent to humans.

In a further embodiment of the present invention, cultured neurons are used for the diagnosis of central nervous system diseases. Neurons are obtained from an individual suspected of having a central nervous system disease and cultured according to the present invention. The presence or absence of diseased state proteins is then detected. The individual would be diagnosed with the

disease if the proteins are detected. For example, Alzheimer's can be diagnosed by the detection of APPs. Neurons from an individual which are cultured in accor¬ dance with the present invention, are incubated in a calcium salt and then contacted with a calcium ionophore. The presence or absence of APPs is detected using standard methods known in the art.

For purposes of illustrating a preferred embodi¬ ment of the present invention, in the following non- limiting example, cultures of olfactory neurons from individuals without central nervous system diseases and from individuals with Alzheimer's disease were estab¬ lished. It is, however, to be understood that the dis¬ cussion generally applies to established cultures of neurons from individuals with any central nervous system disease.

EXAMPLE Procurement of Olfactory Epithelium Olfactory epithelium was procured from cadavers of healthy individuals and Alzheimer's individuals using a cup shaped curette 6 cm in length and with a curette size of 5 or 7.5 mm (depending on the size of the nasal pas¬ sage) .

One of the two approaches (Anterior or Posterior) was taken to obtain epithelium containing olfactory neurons. In the "Anterior" approach, the curette was advanced into the nasal vestibule. The curette was then placed adjacent to the first nasal turbinate (concha) encountered. The first turbinate was usually encountered after the curette was advanced about 3 or 4 cm into the nasal vestibule.

The turbinate was scraped 3 or 4 times with the curette. Olfactory epithelium, yellower than the sur¬ rounding tissue was obtained. Since the olfactory tissue lies over the bone, the presence of some bone associated with the scrapings is a good sign.

The tissue was placed in modified L-15 transport medium containing polyvinylpyrrolidone-360 200 mg/1,

glutathione 0.79 mg/1, 2-mercaptoethanol 50 mg/1, fetal bovine serum 1%, penicillin 200 U/ml, streptomycin sulfate 200 mcg/ml (all above agents were from Sigma or GIBCO) and fungizone 2.5 ug/ml (Squibb). The tissue was transported on ice. However, the sample was not frozen since freezing kills the tissue.

As an alternative to the modified L-15 transport medium described above, the L-15 transport medium may be modified to contain some or all of the agents described by Kischer et al. [Cytotechnology 2:181-185, (1989)].

Procurement of olfactory epithelium using the "Posterior" approach was somewhat more difficult than with the "Anterior" approach.

The olfactory epithelium lies underneath the cribriform plate and sends axons up through the plate into the olfactory bulb. Therefore, the portion of the cribri¬ form plate immediately underneath the olfactory bulb was removed and the tissue underneath collected. It was not necessary to separate the tissue from the attached bone; however, the dura had to be removed.-

The collected tissue was transported as described above in the "Anterior" procurement approach.

Olfactory Neuron Culturing

The olfactory neurons were grown using the basic method described by Coon et al. [PNAS USA 86:1703-1707,

(1989); see also Ambesi-Impiombato et al. PNAS USA

77:3455-3459, (1980)]. The collected tissue was cut into

1 mmx 1 mm pieces and put under a reconstituted basement membrane preparation available as "matrigel" (Collabora- tive Research Inc.) and kept in Coon's 4506 medium.

Coon's 4506 medium uses modified Ham's F-12 medium as a base. Coon's 4506 is made by first preparing Coon's modified Ham's F-12 to include the amino acids, salts and minerals described below in the Table 1. The modified Ham's F-12 is then further modified by including the items listed below in Table 2 to generate Coon's 4506 medium.

Table 1: Coon's Modified Ham's F-12 - neuroblast formulation**

Component grams per liter

L-Alanine 0.018 L-Arginine HCl 0.420

L-Asparagiane Anhyd 0.030

L-Aspartic Acid 0.026

L-Cysteine HC1-H ₂ 0 0.070

L-Glutamic Acid 0.030 L-Glutamine 0.290

Glycine 0.160

L-Histidine HC1-H ₂ 0 0.042

L-Isoleucine 0.008

L-Leucine 0.026 L-Lysine HCl 0.073

L-Methionine 0.009

L-Phenylalanine 0.010

L-Proline 0.070

L-Serine 0.021 L-Threonine 0.024

L-Tryptophan 0.004

L-Tyronsine 2Na 0.016

L-Valine 0.023

D-Glucose 2.00 Biotin 0.00007

D-Ca Pantothenate 0.0005

Choline Chloride 0.014

Folic Acid 0.001

Myo-inositol 0.036 Niacinamide 0.00004

Pyridoxine HCl 0.00006

Riboflavin 0.00004

Thiamine HCl 0.00029

Component grams per liter Vitamin B-12 0.0014

Putrescine 2HC1 0.0003

Na Pyruvate 0.220

Na Hypoxanthine 0.0047

Thymidine 0.0007

L-Ascorbic Acid 0.045

Linoleic Acid 0.00009

Lipoic Acid 0.0002

Phenol Red 0.0012

Sodium Chloride 7.530

Potassium Chloride 0.230**

Sodium Phosphate Dibasic Anhyd 0.135 or as - 7H ₂ 0 0.250

Potassium Phosphate Monobasic 0.068

Magnesium Chloride - 6H ₂ 0 0.081**

Magnesium Chloride - 7H ₂ 0 0.025**

Calcium Chloride 0.0**

Cupric Sulfate - 6H ₂ 0 0.000002

Ferrous Sulfate - 7H ₂ 0 0.0008

Zinc Sulfate - 7H ₂ 0 0.000144

Table 2: Coon's 4506 Medium

MODIFIED F12 (mF12. neuroblast formulation) [Ham's F12 with the following changes:]

2 times the concentration of AMINO ACIDS,

PYRUVATE

ASCORBATE 45 μg/ml

HANK'S SALTS in place of those specified by Ham Hank's formulation further modified so as to contain

Mg++-0.48mM

NO ADDED Ca++ (Final < 0.1 mM from extracts)

KC1 reduced 0.230 mg/ml FOLIC ACID 0.001 mg/ml

HYPOXANTHINE 0.0047mg/ml

THYMIDINE 0.0007mg/ml

GLUCOSE 2 mg/ml

GALACTOSE 0.5 mg/ml NO LINOLEIC ACID

ADDITIVES to make 4506 from mF12 base:

FETAL BOVINE SERUM (GIBCO) 60 %

TRANSFERRIN (HUMAN) 5 μg/ml

INSULIN (Na+) 1 μg/ml HYDROCORTISONE 3.5 ng/ l

SELENOUS ACID 2.5 ng/ml

THYROXINE T ₃ 40 pg/ml

GENTAMYCIN SO _« (GIBCO) 50 μg/ml

EXTRACTS (final concentrations) : BOVINE HYPOTHALAMUS 150 μg protein/ml

BOVINE pituitaryMUS 50 μg protein/ml

CULTURES made in droplets or on plates coated with:

"BASEMENT MEMBRANE" ~150 μg protein/ml (e.g. "Martrigel" Collaborative Research, Inc.)

The above concentrations of Mg++, Ca++, KCl, transferrin, insulin, hydrocortisone, selenous acid, and gentamycin S0 ₄ can, advantageously, be varied by +_ 10%; the above concentrations of ascorbate, folic acid, hypo- xanthine, thymidine, glucose, galactose, fetal bovine serum, T ₃ , bovine extracts and basement membrane can, advantageously, be varied by ± 50%. Variations in the preferred ranges indicated above, as one skilled in the art will appreciate, may be acceptable and/or advantageous depending, for example, on the cell type to be grown. The optimal concentrations can readily be determined.

After several weeks of culture, neurons began to grow. A variable number of tissue pieces, between 10-100% grew out neurons. Neuronal cultures were selected based on the morphology of the cells. Other types of cells that grew out were epithelial, glandular and a spindly type. (See Figure 1). The basement membrane functioned to inhibit growth of other cell types and promote neuronal growth. The neurons were collected as in Coon et al. [PNAS

USA 86:1703-1707, (1989)] and grown in cell culture dishes coated with basement membrane. Dishes were coated with basement membrane by spreading cold basement membrane on

the dish and then leaving the dish at 37°C for at least 10-20 minutes. The Coon's 4506 Medium was changed twice a week. Cells were not allowed to remain confluent for more than 2 days. The neurons were harvested from the dishes by treating the neuron cultures with a protease solution, Dispase (Boehringer-Mannheim, Indianapolis) for 1 hr at 37°C. The medium containing the detached cells was spun down at 1000 rpm for 10 min, the supernatant removed and then the cells were resuspended in appropriate medium. Cells were always placed onto plates coated with basement membrane solution.

For storage, cells were in Coon's 4506 medium containing 10% dimethylsulfoxide. Cells were frozen down under liquid nitrogen. Clonal colonies of neurons were also obtained by diluting harvested neurons in Coon's 4506, growing them on basement membrane coated dishes, isolating individual colonies using cloning cylinders (BellCo) and then har¬ vesting individual colonies as described above. Coon's 4506 medium was required for initial growth of the neurons. Once established, the culture may be able to be maintained using other mediums such as Keratinocyte Growth Medium (Clonetics, San Diego) instead of the Coon's 4506. Identification of Neuronal Cells

Neuronal cells were identified using immunocyto- chemistry and Western blot methodologies well known in the art. The cells were shown to express several neuron- specific proteins: Neuron Specific Enolase, Neurofila ent (68 kD form) and Tau Protein (see Figure 2, Columns A-D) . Column A of Fig. 2 is a blank (negative control) . In Column B of Fig. 2 the arrows point to the Western Blot of Neuron Specific Enolase (Lanes 11 and 12 are the cells and Lane 13 is the brain tissue) . Column C of Fig. 2 shows the circled area with the arrow representing Tau protein, another Neuron Specific Protein.

From a tissue sample containing only neurons, a pure neuron population was grown out without the aid of

SUBSTITUTESHEET

13a the basement membrane. The Coon's 4506 had an increased amount of fetal bovine serum (20%) and culture growth was slower.

Human olfactory neurons strains 1402 and 90-1 were deposited on March 6, 1990 under the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn

SUBSTITUTE SHEET

14

Drive, Rockville, Maryland 20852 under Accession Nos.

ATCC CRL 10366 and ATCC CRL 10367, respectively.

Detection of Altered Amyloid Precursor Protein (APP) Metabolism To detect APP metabolism and identify Alzheimer's diseased olfactory neurons, olfactory neurons from patients with Alzheimer's disease were cultured as described above. The cultured neurons were preincubated for 24 hours in a final concentration of 3mM CaCl ₂ . While CaCl ₂ was used, it is believed other calcium salts may also be utilized at concentrations as low as lmM.

After preincubation, the calcium ionophore CA23187 was added to the neuron culture at a final concentration of 100 nM. The ionophore was added to elevate intracellu- lar calcium in order to activate calcium dependent pro¬ teins. It is believed concentrations of ionophore as low as lOnM may be effective. The culture was then incubated for 24 hours. Some effect was seen after 1-4 hours, however, a greater effect was seen after 24 hours of incubation.

The incubated neurons were homogenized and a standard immunoblot assay was preformed using anti-A-4 antibody (Boehringer Mannheim, Indianapolis, IN). (Anti- A4 recognizes the all region common to all APPs.) 60 μg per lane were electrophoresized on a 8% or 10% polyacryl- amide gel and transferred to nitrocellulose. Nonspecific binding was blocked with bovine serum albumin prior to treatment with anti-A4 antibody. A phosphatase coupled avidin-biotin conjugate kit (Vector Labs, Burlingame, CA) to visualize APP species. The Alzheimer's disease spe¬ cific reactivity, that is anti-APP reactivity, was seen at

100 + 10 KD. (see Figure 3).

* * * * *

The entire contents of all references cited hereinabove are hereby incorporated by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading

15 of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.