# Patent

## Title
Neurodegeneration mutants, method for identifying same, and method for screening neuroprotective agents

## Abstract

A method for obtaining a Drosophila neurodegeneration mutant includes the steps of selecting at least one Drosophila mutant having an aberrant phenotype selected from the group consisting of temperature-sensitive paralysis and bang-sensitive paralysis; and screening the at least one selected Drosophila mutant for age-dependent neurodegeneration. Neurodegeneration mutants are obtained in the method and causal mutations are characterized. The neurodegeneration mutants can be used in screening methods to identify putative neuroprotective agents.

## Background
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with United States Government support awarded by the following agency: NIH Grant No. NS15390. The United States has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATION
Not applicable.


BACKGROUND OF THE INVENTION
The cellular and molecular mechanisms underlying age-dependent neurodegeneration seen in metazoans from worms to humans are poorly understood. Neurodegeneration is largely genetic in origin and often results from a single gene defect. (reviewed in FORTINI and BONINI 2000; HEINTZ and ZOGHBI 2000; FEANY 2000; FORMAN et al. 2000; GOEDERT 2001; SIPIONE and CATTANEO 2001; MACCIONI et al. 2001). Human neurodegenerative illnesses, such as Amyotropic Lateral Sclerosis (ALS), Huntington disease, Parkinson disease, and Alzheimer disease are characterized by progressive behavioral deficits, premature death and, in some cases, profound cognitive impairment. Onset of the symptoms of these diseases correlates with the appearance of neuropathology. Mutations that underlie some forms of these diseases are known, but a general understanding of the molecular mechanisms required for maintaining neuronal viability are not understood.
A great demand exists for therapeutic interventions for neurodegenerative diseases, particularly for administration to elderly subjects. Notwithstanding the great demand, few if any drugs are available to reduce age-related neurodegeneration. At the heart of this lack of therapeutic agents is the lack of adequate screening assays for novel therapeutic interventions. Typically, neuroblast-derived cell lines in culture are exposed to putative agents and agents that extend the life in culture are selected for further investigation. What is missing is an efficient in vivo technology for use as a primary screen or for confirming effectiveness of putative neuroprotective agents.
Human neurodegenerative conditions can be modeled in Drosophila. In some cases, human proteins, such as alpha-synuclein and tau, are expressed in Drosophila and cause neurodegenerative syndromes having phenotypic properties similar to those of Parkinson and Alzheimer diseases, respectively. Likewise, flies expressing human Huntingtin containing expanded triplet repeats develop neuropathological defects reminiscent of human Huntington disease. Using this model system, various suppressor mutations have been isolated. It has further been determined that onset of neurodegeneration in flies can be suppressed by overexpressing human hsp70. Accordingly, it appears that Drosophila can be instrumental in uncovering key mechanisms of general significance in the field of neurodegenerative disorders.
Additionally, Drosophila have been screened for single gene mutations that cause neurodegeneration. Mutants such as drop dead, swiss cheese, eggroll, spongecake, and bubblegum have moderate to markedly reduced lifespans and associated neuropathology including vacuolization and accumulation of multi-lamellar cell bodies. These neuropathologies are similar to those seen in patients having Tay-Sachs and Creutzfeldt-Jakob diseases.
Still, the number of neurodegeneration mutants is quite small and additional neurodegeneration mutants are of interest. Such mutants not only help in deciphering basic neurodegeneration biology, but can also serve as convenient and inexpensive models both for genetic therapies and for screening putative neuroprotective agents. It is, of course, difficult to ascertain which strains of Drosophila exhibit neurodegeneration. It is impractical to screen all strains and mutants for neurodegeneration and the art lacks a principled basis upon which one would select candidates a priori. Prior efforts have examined flies on the basis of a defect in phototaxis or reduced life span. (HEISENBERG and BOHL, 1979; HEISENBERG 1979; COOMBE and HEISENBERG 1986; BUCHANAN and BENZER 1993; MIN and BENZER 1997; KRETZSCHMAR et al. 1997). For example, Min and Benzer, 1997 examined five thousand mutagenized lines and isolated sixty mutant lines having reduced lifespan. Of the sixty mutants, two (0.4% of the mutagenized lines examined) exhibited neurodegeneration. Screening for mutant lines having reduced life span presents at least two inherent disadvantages, namely the substantial time required to ascertain a shortened life span and the low incidence in such mutants of neurodegeneration. A more targeted and efficient approach to screening for neurodegeneration mutants in Drosophila is desired.
Previous studies have established a connection between neuronal dysfunction and neurodegeneration in some species. Some neurodegenerative mutants affect genes that encode ion channels and neurotransmitter receptors. Weaver (wv), lurcher (Lc), and tottering (tg) were identified in mice on the basis of locomotor behavior defects and contain mutations in genes that encode ion channels and neurotransmitter receptors. Studies of these mutants have demonstrated an important connection between aberrant neuronal signaling properties and neurodegeneration (MURTOMAKI et al. 1995; NORMAN et al. 1995; FLETCHER et al. 1996; ZUO et al. 1997). The connection is also established in the worm sensory system (HALL et al. 1997). An indirect connection between neurodegeneration and ion channels is seen in Drosophila dADAR mutants which exhibit extensive neurodegeneration arising from lack of an enzyme essential for adenosine to inosine type editing of pre-mRNAs that encode several Drosophila ion channels. Notably, dADAR null mutants undergo extensive neurodegeneration (PALLADINO et al. 2000a).
This application also describes various mutations in the alpha subunit of Na+/K+ ATPase pumps (sodium pumps) that asymmetrically distribute Na+ and K+ ions to form ion gradients across the plasma membrane of cells. These ion gradients determine the membrane resting potential and excitability of cells and drive many important secondary processes. Without such ion gradients, many essential functions, including electrical signaling in the nervous system, are not possible. Many sodium pump isozymes exist, are highly conserved evolutionarily, and are widely expressed in animal tissues. In neurons, sodium pumps generate and maintain the membrane potential after extensive Na+ influx enabling continued generation of action potentials. Not surprisingly, sodium pumps are extensively regulated in vivo (reviewed in THERIN and BOLSTEIN, 2000).
Sodium pumps have at least two essential subunits, alpha and beta. The alpha subunit of the Drosophila Na+/K+ ATPase (ATPalpha) is a large protein (>110 kDa) with multiple transmembrane domains and an ATP-dependent catalytic activity. A version of Drosophila ATPalpha is available in Genbank at Accession No. XP-081160, presented herein as SEQ ID NO:1. Mutations and reversions described herein are defined relative to the ATPalpha amino acid sequence disclosed in Accession No. XP-081160 and are not separately presented. It is understood that the skilled artisan can readily understand the complete sequences of mutants and reversions from the information presented in the specification. The beta subunit has a single transmembrane domain and may be involved in pump maturation, membrane localization and functional properties of Na+/K+ ATPases.
Studies characterizing the functions and importance of Na+/K+ ATPase proteins in vivo in other animals are limited but suggest that normal neural development and maintenance requires proper Na+/K+ ATPase function. One study of Na+/K+ ATPase loss-of-function eat-6 mutations in the nematode established a link between pharyngeal function and sodium pump activity. Null mutations of the mouse Na+/K+ ATPase beta2 subunit cause neural cell degeneration, apoptotic photoreceptor cell death, and death late in development.
The importance of Na+/K+ ATPase function has also been suggested by widespread expression in metazoan tissues, striking evolutionary conservation, and involvement in many essential processes including nutrient absorption, nephritic function and signaling in the nervous system. Many studies suggest a pathophysiological connection between the biochemical function of these important proteins and human neural diseases including bipolar disorder, seizures and neurodegenerative conditions, namely spongiform encephalopathies, with manifestations similar to those caused by prion diseases, namely Kuru, Crutzfeld-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome (reviewed by MOBASHERI et al, 2000).
Na+/K+ ATPase function is also implicated in cardiac hypertrophy, hypertension, renal dysfunction, bipolar mood disorder, and spongiform encephalopathies. Somewhat surprisingly, no direct mutation of Na+/K+ ATPase alpha has been identified as the cause of neural disease.
Among conditional paralytic mutants, mutations are known to cause neuronal dysfunction by disrupting polynucleotides that encode electrical signaling proteins. One bang-sensitive paralytic mutation and several lethal p element insertions have been mapped to ATPalpha (SCHUBIGER, 1994), (FENG, 1997). Additionally, transgenic ATPalpha having specific modifications at a phosphorylation site required during ATP hydrolysis causes bang-sensitive paralysis and, in some cases, death (SUN, 2001).
No association between neurodegeneration and mutations in Na+/K+ ATPase alpha has been reported, although loss of Na+/K+ ATPase function can cause neuropathological effects (reviewed in Beal, 1993; Lees, 1993). These neuropathological effects are seen after administering Na+/K+ ATPase inhibitors or in the presence of mutations that affect Na+/K+ ATPase beta subunits. In addition, inherited defects associated with reduced Na+/K+ ATPase activity have been linked to neonatal status convulsivus, spongiform encephalopathy (RENKAWEK et al, 1992).
BRIEF SUMMARY OF THE INVENTION
A novel method is disclosed for identifying neurodegeneration mutants in Drosophila and specific mutations in the nucleic acid of the neurodegeneration mutants and polypeptides encoded by the mutants. Neurodegeneration mutants are advantageously and efficiently obtained in a selection and screening method, in which a selected collection of temperature-sensitive paralytic mutants or bang-sensitive paralytic mutants are screened for neurodegeneration as described herein. The high incidence of neurodegeneration mutants found among temperature-sensitive paralytic mutants and bang-sensitive paralytic mutants suggests that perturbation of neuronal signaling pathways can contribute to neurodegeneration.
In accord with one aspect of the invention, the inventors herein demonstrate that a collection of mutants exhibiting aberrant behaviors associated with altered or impaired neural function are enriched in mutants that exhibit characteristic neurodegeneration. In a related aspect, therefore, one principle of the invention is the novel appreciation that the task of identifying neurodegeneration mutants is eased appreciably by selecting as a starting material for use in the method of the invention a collection of such behavioral mutants. The skilled artisan will appreciate that the likelihood of identifying a neurodegeneration mutant in the method of the invention increases with an increase in the number of screened behavioral mutants.
In particular, temperature-sensitive paralytic behavioral mutants and bang-sensitive paralytic behavioral mutants are advantageously used as starting materials to be screened in the method of the invention. Mutants to be screened in the method of the invention can include, but need not be limited to mutants carrying non-wild-type ion channel structural genes, ion channel regulatory genes and genes affecting synaptic transmission machinery. Many temperature-sensitive paralytic strains contain mutations in genes that encode ion channels, components of the synaptic machinery, and other proteins required for generating and transmitting electrical signals in the nervous system. (LOUGHNEY et al. 1989; ATKINSON et al. 1991; PALLANCK et al. 1995; TITUS et al. 1997; LITTLETON et al. 1998).
Neurodegeneration mutants identified in the method of the invention are valuable research tools for identifying key proteins and biochemical pathways required for maintenance of neuronal viability. The mutants are also advantageously used to develop novel therapies for avoiding natural senescence and for treating and preventing neurodegenerative disorders in human and non-human animals.
In a working embodiment of the method, mutants were identified at a frequency that compares favorably with a prior large-scale screen of reduced lifespan flies, supra. In the selection step, temperature-sensitive paralytic mutants were identified at a frequency of 0. 1–0.2%. In the screening step, extensive neurodegeneration was observed in about 20% of the lines examined (0.02–0.04% of the mutagenized lines). The approach disclosed herein is preferred over prior methods for isolating neurodegeneration mutants, however, because it is much easier to score paralysis than lifespan and because reduced lifespan is not a characteristic of all mutants subject to age-related neurodegeneration.
Among the neurodegeneration mutants identified in the method are several novel dominant and recessive conditional temperature-sensitive and bang-sensitive paralytic mutations in Drosophila ATPalpha, having characteristic age-dependent behavioral abnormalities that can include conditional paralysis and a profound bursting physiological hyperexcitability defect. “Bursting” is described in Kasbekar, D. P. et al, “enhancer of seizure: A New Genetic Locus in Drosophila melanogaster Defined by Interactions with Temperature-Sensitive Paralytic Mutations,” Genetics 116:423–431 (1987), incorporated by reference herein as if set forth in its entirety. Additionally, flies containing dominant ATPalpha mutant alleles exhibit a characteristic massive, age-dependent neurodegeneration. Maintenance of neuronal viability depends on normal sodium pump activity; aberrant ATPalpha function results in seizures and marked spongiform neuropathology.
In particular, the invention relates in part to a novel ATPalpha polypeptide having an amino acid sequence as disclosed in Genbank Accession No. XP-081160 modified relative to that sequence so as to have a substitution mutation affecting residue 981, residue 982, or both residues. Also, the invention relates to a polynucleotide that encodes any of the polypeptides of the invention. A polynucleotide of the invention can be provided on a cloning vector or expression vector. In the latter case, the vector can also comprise an upstream heterologous promoter and any other transcription or translation element advantageously employed in vivo or in vitro to obtain a polypeptide of the invention.
Other objects, features and advantages of the present invention will become apparent upon consideration of the following detailed description of the invention.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Not applicable.



DETAILED DESCRIPTION OF THE INVENTION
Neurodegeneration is enriched among various mutants characterized as having aberrant behaviors likely to be associated with altered or impaired neural function, namely temperature-sensitive paralytic mutants and bang-sensitive mutants. Numerous genes that encode ion channels and proteins required for synaptic transmission have been identified in temperature-sensitive paralytic mutants. In a method according to the invention, temperature-sensitive paralytic mutants or bang-sensitive mutants, or combinations thereof, are subjected to a histological screen, as described elsewhere herein, and those strains of mutant Drosophila exhibiting neurodegeneration on histological examination are selected for detailed phenotypic and molecular characterization or for use is subsequent methods for screening putative neuroprotective agents. The method provides an efficient means for targeted identification and collection of neurodegeneration mutants.
Those skilled in the art are familiar with methods for obtaining temperature-sensitive paralytic mutant flies strains, which can be isolated de novo according, e.g., to the method of Wu, C. F., et al., “A Drosophila mutant with a temperature-sensitive block in nerve conduction,” P.N.A.S. USA 75:4047–4051 (1978), incorporated herein by reference as if set forth in its entirety. An adult or larval mutant is a “temperature-sensitive paralytic mutant” if it becomes paralyzed, ataxic or severely uncoordinated in less than 10 minutes when exposed to temperatures above 28° C. The behavioral phenotypes are reversible and the flies or larvae recover normal or nearly normal locomotor activity with a time course that varies from a few seconds to several hours depending upon the strain. One can decide whether a mutant is paralyzed, ataxic or severely uncoordinated by direct observation or by scoring performance on any one or, preferably more than one, of the running, climbing and flying tests described below in the Examples. A suitable strain that can function as a wild type control that does not become paralyzed at 37–38° C. is the Canton-S strain.
Those skilled in the art are familiar with methods for obtaining bang-sensitive paralytic mutant flies strains, which can be isolated de novo according, e.g., to the method of Grigliatti, T. A. et al., Molec. Gen. Genet. 120, 107–114 (1973), incorporated herein by reference as if set forth in its entirety. A mutant is a “bang-sensitive paralytic mutant” if it becomes paralyzed, ataxic or severely uncoordinated in less than 10 minutes when subjected to mechanical stress. Sufficient mechanical stress is provided, for example, by a vortex at highest speed for 10–20 seconds. The utility of the method does not depend upon the commercial availability of the starting mutants, but rather the use of same without regard to the means by which the strains are obtained.
In the method, the temperature-sensitive paralytic mutants or the bang-sensitive mutants, or both, are aged to an age at which neurodegeneration can be assessed. It is convenient, but not essential, to evaluate the mutant strains for neurodegeneration in the method at the midpoint age of the strain's survival curve which can be determined as described below. Neurodegeneration in the intact fly cannot be observed by the naked eye, but can be assessed by known histological methods, as detailed below, or by other diagnostic methods.
For a more thorough assessment of the neurodegeneration mutants revealed in the screen, the mutants can be compared to wild type flies in behavioral assays or electrophysiological techniques such as those performed at the larval neuromuscular junction, electroretinogram (ERGs), or those performed on adult flight muscles (Wu and Ganetzky, 1992). Synaptic transmission in the visual pathway can be measured by electroretinogram (ERG), an extracellular recording from the compound eye, that measures light-induced depolarization of photoreceptors as well as the synaptic-mediated responses of second-order neurons in the visual system (Hotta and Benzer, 1969; Pak et al., 1969). The ERG includes a component maintained during the entire light flash that corresponds to the light-dependent depolarization of the photoreceptor cells as well as on- and off-transients that appear at the beginning and end of a light flash respectively. The on- and off-transients represent responses from the second-order neurons in the lamina. If synaptic transmission between photoreceptor cells and laminar neurons is blocked, the on- and off-transients are preferentially lost.
The present invention also relates to various temperature-sensitive paralytic and bang-sensitive paralytic Drosophila mutants, preferably single point mutants, for which one inventive aspect is the recognition that the mutants exhibit substantial neurodegeneration as disclosed herein.
Additionally, the mutants identified in the method are advantageously used as targets for screening putative therapeutic agents for reversing the neurodegeneration associated with the mutants. In such methods, the mutant flies are exposed to a neuroprotecting amount of a putative neuroprotective agent. Neurodegeneration is assessed at or after the time at which neurodegeneration would be observed in flies untreated with the putative neuroprotective agent. The skilled artisan can readily perform the preparatory trials underlying the method to determine, for each strain tested, the amount of the agent to use, the duration of exposure to the agent, and the time at which neurodegeneration or neuroprotection should be assessed. The agent can be tested by direct injection into fly hemolymph using known methods. For example, using a pulled glass needle and a small volume injection apparatus such as the Drummond Nanoject (cat. no. 3-00-302-x) or related apparatus, a compound can be injected into the abdomen in a phosphate buffered saline. The procedure can be done without affecting the lifespans of the flies. It will also be appreciated by the skilled artisan that the screening need not be performed in flies, but can be performed in other animal systems in which neurodegeneration can be assessed.
The methods and mutants disclosed herein also find application in the field of identifying polynucleotide sequences in Drosophila that are associated with neurodegeneration and which have homologous sequences in vertebrate animals, especially mammals, and particularly in humans. Sequences in such animals identified as being associated with neurodegeneration can be isolated and introduced by transgenic methods into Drosophila or other model organisms, whereby the ability to employ neuroprotective agents to alter neurodegenerative outcomes can be assessed.
The invention will be more fully understood upon consideration of the following non-limiting Examples.
EXAMPLES
Example 1
In the selection step of the method of the invention, a set of mutant Drosophila strains were selected for a reversible, temperature-sensitive paralytic behavioral phenotype. The mutants primarily comprise independent, chemically induced, temperature-sensitive (TS) paralytic mutations. The mutants were isolated primarily in standard screens of the major chromosomes following mutagenesis with ethylmethane sulfonate (EMS). Flies were cultured on standard cornmeal, molasses agar medium at room temperature (21–23° C.). Flies and larvae were placed at 37–38° C. and flies or larvae that became paralyzed, ataxic, or severely uncoordinated in less than five minutes at the elevated temperature were retained for further study. The flies and larvae recovered normal or nearly normal locomotor activity within a few seconds to several hours, depending on the mutant strain. Many of the mutations affect genes that encode ion channels, components of the synaptic machinery, and other proteins required for the proper generation and transmission of electrical signals in the nervous system (WU and GANETZKY 1992; LITTLETON et al. 1999).
In the screening step of the method, the selected set of mutant strains were screened for age-dependent neurodegeneration by histological analysis. Mutant and control strains were screened as larvae, as young adults, and at the midpoints of their respective adult lifespan curves. The lifespan curves were determined as follows. Flies were raised to adulthood at room temperature (20–25° C.) and newly eclosed flies were placed in vials at low density (10–20 flies per vial) and incubated at 28° C. Males and females were kept in separate vials. Flies were transferred daily into fresh vials to minimize deaths caused by microbial infection or becoming stuck in the medium. The number of surviving flies was recorded daily. Survival curves were generated by plotting the percent of surviving flies as a function of time (in days). Surviving flies were removed for histological analysis and flies that suffered incidental deaths were excluded from the calculation. Statistical analyses were performed using Student's t-Tests.
Larvae, young, and aged adult flies (wild type controls and mutants) were screened for central nervous system (CNS) neurodegeneration, as indicated by a loss of tissue or vacuolar appearance of neural tissue of the brain, ganglia or eye tissue. To accomplish the screen in young and aged flies, heads were removed and were fixed in freshly prepared Carnoy's fixative at room temperature for 4–12 hours and processed into paraffin using standard histological procedures. Heads were embedded to obtain sections. Serial, frontal 4 uM sections were obtained, stained with hematoxylin and eosin and examined under a light microscope (n>25, each genotype) for gross neuropathology. For ventral ganglia examination the bodies were fixed similar in Carnoy's and processed into paraffin using standard histological procedures. Serial, saggital 4 uM sections were obtained, stained with hematoxylin and eosin and examined under a light microscope (n>25, each genotype) for gross neuropathology. For larvae, the entire CNS was removed, fixed in Bouin's, and processed into JB-4 embedding medium (Polysciences). Horizontal, 3 uM sections were obtained and stained with hematoxylin and eosin and then examined under a microscope (n>12, each genotype).
Among approximately 80 different temperature-sensitive mutant lines examined, 10 exhibited extensive CNS neurodegeneration. Five additional mutants displayed significant, but less dramatic, neurodegeneration. Additionally 6 control strains were examined for frequency and types of pathology that appear in aged wild type animals. The high frequency of neurodegeneration mutants among this collection demonstrates the utility of this approach.
One mutant revealed in the screen, vacuous (vacu), incurred extensive neuropathology. vacu is a recessive mutation generated on an st-marked third chromosome that was originally identified on the basis of larval paralysis at 38° C. Mutant larvae placed on a heated agar slab immediately cease crawling; in contrast, wild type larvae crawl vigorously at 38° C. Paralysis of vacu larvae reverses rapidly upon return to permissive temperatures. The lifespan of vacu at 28° C. is only about half that of controls—both fifty percent and maximal age are reduced in vacu animals (19.3 and 41.5 days) versus wild type (43.6 and 50.7) (p<0.001). The reduced lifespan of vacu is characteristic of this mutant and is not a phenotype shared by all temperature-sensitive paralytic mutants.
Histological analysis of frontal sections of heads from vacu adults revealed readily observable neurodegeneration with widespread and massive loss of neural tissue in the neuropil of the central brain and in the optic lobes (evident as the appearance of apparent vacuolization throughout these regions). Neuropathology is highly-penetrant in aged vacu animals: every animal tested exhibited significant gross pathology (n>60 animals). In contrast, aged control animals never exhibited significant gross pathology and it was rare to observe even a single vacuolar clearing (n>50 animals). Similar neurodegeneration was also readily observed in sagittal sections of the thoracic ganglion of vacu adults. The thoracic ganglion pathology is also highly-penetrant in vacu animals.
vacu neurodegeneration is progressive and varies as a function of developmental stage and age. No obvious evidence of neurodegeneration was observed in the third instar larval CNS, even though vacu larvae showed a strong behavioral defect. Likewise, young vacu adults did not exhibit neurodegenerative phenotypes within 36 hours post-eclosion. The age-dependent decline in behavior that correlates with the onset of observable neuropathology is similar to what is seen in progressive neurodegenerative diseases in humans.
Behavioral defects in the vacu adult flies also become more apparent with increasing age. Four groups of flies, corresponding to “young” and “aged” populations of wild type and vacu flies, were tested in the behavioral assays. Ten samples of 20 flies each (n=200 for each group) were collected for each of these four groups. Young flies were collected within 24 hours of eclosion and aged for 24–48 hours at 28° C. before testing. Aged flies were also collected within 24 hours of eclosion and maintained at 28° C. for 11 days. Each sample of flies was subjected to the same set of behavioral assays in the same order to test running, climbing, and flight abilities.
In a first behavioral assay (running), flies were placed at one end of a darkened, horizontal tube and allowed to run toward a light source at the other end. Eight flies were randomly selected from each sample and placed in a 10 mL glass pipette, sealing both ends with wax film to prevent escape (n=80 animals per group). One end of the pipette (20 cm) was darkened by placing it inside a dark-colored foam block. With the pipette secured horizontally in the foam, light from a fiber optic lamp was shined directly into the exposed tip of the pipette. The lamp was placed as close to the tip of the pipette as possible. For each trial, the flies were knocked to the foam-covered end of the pipette then returned to the horizontal test position. The time required for the first six flies of each sample to enter the light-exposed portion of the pipette was measured in seconds. Four trials were completed for each sample. Each individual fly was given a score and these scores were then averaged for each sample for statistical analysis using Student's t-test.
Young wild type adults typically required less than 30 seconds (s) to run the length of the tube. Young vacu adults were somewhat slower than the controls, although they still ran the length of the tube in only about 45 s. Although newly eclosed vacu adults did not show any overt behavioral abnormalities other than generally sluggish locomotor activity, either at 20° C.–22° C. or at 37–38° C., as they age vacu adults differ markedly from wild type in viability and locomotor activity. While both wild type and vacu adults showed an age-dependent decrement in this assay (p<0.001), the decline was much more severe for vacu (7.5 versus 5.1 fold increase). Aged wild type adults ran the tube in about 150 s, whereas aged vacu adults required around 300 s. Aged wild type performed significantly better than vacu animals (p<0.001).
In a second behavioral assay (climbing), ten flies were randomly selected from each sample and placed in a 250 mL glass graduated cylinder, sealed at the top with wax film to prevent escape (n=100 animals per group). A fiber optic lamp illuminated the cylinder from the top. The flies were gently knocked to the bottom of the cylinder and flies climbed up a vertical tube against gravity toward the light source. The time required for 50% of the flies to cross a line 17.5 cm from the bottom of the tube was determined. Four trials were completed for each sample. Times for each sample were averaged for statistical analysis using Student's t test. Young wild type adults reached this point within 5–10s. Wild type flies showed a small age-dependent decrease in behavior (p<0.001), requiring about 30–35s to cross the line. In contrast, vacu adults, both young and old, performed very poorly in this assay. Neither group of vacu adults completed the task within the total five minute period allotted. Because even young vacu adults behaved so poorly in this assay, it was not possible to assess whether vertical climbing ability declined further with age.
In a third behavioral assay (flight), the assay was performed essentially as in Benzer (1973) and in Elkins et al. (1986). Flies were dropped into the top of a 500-mL glass graduated cylinder through a glass funnel whose end reached the 500-mL mark (n=200 per group). The inside surface of the cylinder was coated with paraffin oil causing flies to become stuck where they strike the wall. The strongest fliers initiate flight immediately and become stuck near the top of the cylinder. The weaker fliers fall farther and become stuck near the bottom of the cylinder. The vertical distribution of each group of flies over the length of the cylinder was determined to measure flying ability. These scores were then averaged for each group of flies for statistical analysis using Student's t-test.
Both wild type and vacu flies show an age-dependent decrease in flight ability but vacu flies of both age groups perform markedly worse than wild type and the decline in flight ability with age is more severe for vacu.
The larval paralytic phenotype of vacu was mapped to between st (3–44.0) and Sb (3-58.2) by recombination analysis. Among 80 recombinants recovered between st and Sb, 24 were between st and vacu, placing vacu approximately 4.3 map units to the right of st. The mapping was refined using existing deletions in the relevant interval to determine the cytological location of vacu. Df(3R)by10(85D8-12; 85E7-F1), Df(3R)by62 (85D11-14; 85F6), and Df(3R)by77 (85D8-12; 86B4) all uncovered the vacu paralytic phenotype, whereas Df(3R)by416 (85D10-12; 85E1-2) did not. These results suggest that the vacu mutation is either in the 85D8-85D 10 interval or in the interval between 85E 1-3 and 85E7-F 1. No other deletions are currently available to resolve these two possibilities.
Anomalous electrical activity in the mutant suggests that loss of vacu function may result in neuronal hyperexcitability. Electroretinograms (ERGs) were recorded essentially as described (PAK et al. 1969; HOTTA and BENZER 1969). Briefly, flies were anesthetized with CO2, their wings and anterior legs were surgically removed, the flies were immobilized in plasticine, and were allowed to recover for 15 minutes. A temperature-controlled stage was used with a temperature probe inserted into the plasticine adjacent to the animal. Glass recording and reference electrodes filled with 3M KCl were placed in the cornea and thorax, respectively. Following dark adaptation (5 minutes), photoreceptor responses to brief light exposures were recorded. Traces were amplified using an Axopatch 1-D amplifier in current clamp mode (clamping at zero) and recorded using Clampex 6.0.3 software (Axon Instruments). Current traces were filtered at 1 kHz and consecutive traces are reported from representative animals (n>6 for each genotype). Recordings were taken at temperatures between 20° C. and 37° C. in the following order: 20° C., 34° C., 37° C. and 22° C.
Sporadic depolarization events were observed at all temperatures in vacu animals. Bursts of apparent electrical activity are present at 34° C. and 37° C. in vacu animals but not in wild type animals. The ERGs of adult vacu flies appeared relatively normal both at 20° C. and at 37° C., despite the noted behavioral deficits. However, recurrent spikes are apparent in the ERG trace at 20° C. before, during, and after the light exposure. At 34° C.–37° C., more prominent sustained bursts of apparent activity are observed both preceding and following the light flash. Upon return to 20° C., the more extreme bursting activity disappears but the small recurrent spikes persist. We believe that this excessive activity originates in the ventral ganglion and is being detected by the ERG ground electrode, which is inserted into the thorax. Similar bursting activity has been previously observed in mutants such as seizure (sei), which are known to cause neuronal membrane hyperexcitability (ELKINS and GANETZKY 1990; TITUS et al. 1997) as well as in some bang-sensitive mutants, and such activity has been interpreted as neural hyperactivity and physiological seizures (PAVLIDIS and TANOUYE 1995; KUEBLER and TANOUYE 2000; KUEBLER et al. 2001). The massive degeneration seen in the optic system appears to be light-independent.
Accordingly, it is shown in this Example that a mutant selected on the basis of its temperature-sensitive paralytic phenotype was identified as a neurodegeneration mutant in accord with the method of the invention. Further, the neurodegeneration mutant was characterized by behavioral and synaptic transmission assays.
Example 2
In extensive screens for mutations that cause neuronal dysfunction in Drosophila we identified several dominant mutations in Drosophila were identified that result in behavioral abnormalities. Two mutants were independently isolated, temperature-sensitive (TS) paralytic mutations and a third was isolated as a bang-sensitive paralytic mutation. All three mutations are homozygous lethal early in development. In accord with the method of the invention, prior selection of temperature-sensitive paralytic mutants and/or bang-sensitive paralytic mutants sensitizes a subsequent screen for neurodegeneration mutants. Mutants DTS1 (ATPalphaDTS1) and DTS2 (ATPalphaDTS2), described below, exhibit neurodegeneration.
Fly stocks were maintained on standard cornmeal, molasses, agar media at 22–28° C. Wild type and control flies refer to Canton S, unless otherwise specified. The DTS1 mutation was obtained in an EMS mutagenesis of Canton S in a screen for dominant temperature-sensitive paralytic mutations. DTS2 was obtained in a similar manner, although cn,bw animals were mutagenized. Upon exposure to 37–38° C., DTS1/+ and DTS2/+ adults become completely paralyzed within 10–30 seconds except for minor twitching of the tarsi. Following a three-minute exposure to the restrictive temperature, the flies regain the ability to stand after one to two minutes at the permissive temperature (<30° C.) and require another several minutes before they begin to walk. Even without exposure to elevated temperatures, both DTS1/+ and DTS2/+ heterozygotes appear to be somewhat sluggish and do not walk or climb as rapidly as wild type flies. Homozygotes for DTS2 and DTS1 die early in development. Mutants DTS1 and DTS2 also manifest a novel temperature-dependent bang-sensitive phenotype. Assays of bang sensitivity and temperature sensitivity were performed as described (GANETZKY, 1982; WU, 1978). When stocks of these flies are maintained at 20–22° C. and tested at the same temperature, they show no obvious bang-sensitivity. However, when the stocks are maintained at 28° C., DTS2/+ and DTS1/+ flies are viable but show bang-sensitive paralysis lasting for 5–30 seconds when tested at room temperature, even when the flies are allowed to accommodate to the temperature shift for several hours. Revertants of DTS1 and DTS2, designated DTS1R1, DTS1R2, DTS2R1, DTS2R2 and DTS2R3, were generated in a screen of gamma ray treated mutants that were no longer TS paralytic.
Mutant H64 was obtained from an ENU mutagenesis of a roe, pp stock. H64 was originally identified on the basis of its bang-sensitive phenotype, and appeared to be an autosomal dominant mutant. When maintained as heterozygotes over the TM6B balancer chromosome marked with Tb, no non-Tb larvae were observed, indicating that H64 homozygotes are lethal prior to the second larval instar. When tested for bang-sensitivity, H64/TM6 adults become completely paralyzed for 10–35 seconds. Upon recovery from paralysis, up to five minutes of additional time is required before these animals regain full activity. These mutants show no evidence of temperature-sensitive paralysis at 37–38° C. but even at 20–22° C. when not subjected to mechanical stimulation, they are often sluggish and spend most of their time on the bottom of the vial.
Complementation tests for recessive lethality among H64, DTS1, and DTS2, in all pairwise combinations (Table 1) shows that all three mutants fail to complement one another, suggesting that they all share lethal mutations of the same gene. The conditional paralytic phenotype of DTS1, DTS2 and H64 was mapped relative to Gl, Sb, H by recombination. All three mutations mapped in the same approximate chromosome location (70.5, 70.3 and 69.5 cM, respectively) and were tightly linked to H. This map position corresponds to the 92D–93B cytological region, which is in the chromosomal region of ATPalpha.







TABLE 1







Viability of existing ATPalpha alleles with the new conditional mutants and their revertants.
















DTS1
DTS1R1
DTS1R2
DTS2
DTS2R1
DTS2R2
DTS2R3
H64



















ATPalpha+
TS, BStd
BS
BS
TS, BStd
BS
mTS, BS
BS
BS


DTS1
l
l
l
l
l
l
l
l


DTS1R1
l
l
l
l
l
l
l
l


DTS1R2
l
l
l
l
l
l
l
l


DTS2
sl, TS
l
l
l
l
l
l
l


DTS2R1
l
l
l
l
l
l
l
l


DTS2R2
l
l
l
l
l
l
l
l


DTS2R3
l
l
l
l
l
l
l
l


H64
l
l
l
l
l
l
l
l


ATPalpha2206
BS, TS
l
l
dl
l
l
l
l


ATPAlpha01453
sl, TS
l
l
l
l
l
l
l





TS = temperature-sensitive paralysis (38 C.),


BS = bang-sensitive paralysis;


BStd = temperature-dependent bang-sensitive paralysis (28 C.);


mTS = modified TS;


l = early developmental lethal;


sl = semi-lethal (5% versus expected 25%);


dl = lethal during pupariation and eclosion.


Note:


ATPAlpha2206/ATPAlpha01453 flies are not bang-sensitive but have reduced viability (10% versus expected 50% of offspring).






DTS1 and DTS2 mutants display a marked age-dependent decrement in locomotor activity. In comparison with age-matched wild-type flies, the mutants become quite sedentary with a premature loss of both walking activity and flight ability.
DTS1 and, to a lesser extent, DTS2 and ATPalpha2206 were determined to be short-lived, whereas H64 has an essentially normal lifespan. Comparisons of the age of 50% survivorship for each genotype demonstrate a significant reduction in lifespan for DTS1, DTS2 and ATPalpha2206 relative to wild type. The time required to reach 50% survivorship for each population was used to compare the lifespans of ATPalpha mutants with control strains TM6, Ubx/+, TM6, Tb/+, and Canton S. Lifespan is significantly reduced in ATPalphaDTS1, ATPalphaDTS2 animals (17 and 27 days) and moderately reduced in ATPalpha2206 (36 days) versus controls (41–45 days) (p<0.001, all comparisons to Canton S). In contrast, the lifespan of ATPalphaH64/+ flies did not differ significantly from Canton S (>0.5).
The reduced lifespan and premature motor activity loss exhibited by both dominant mutants are consistent with the phenotypes of other Drosophila neurodegeneration mutants. To investigate the possibility that these mutants also experienced neurodegeneration, we performed a histological analysis of DTS1/+, and DTS2/+. Wild-type and mutant adults were aged to approximately 50% survival on their respective lifespan curves and histological examination was performed, as in Example 1. Serial frontal sections revealed extensive neuropathology in the brains of all ATPalpha mutants. In DTS2/+ and DTS1/+ animals, vacuolar structures were distributed widely throughout the central brain and optic regions. This phenotype was never observed in wild-type animals which only rarely contained small vacuolar structures. Also, many DTS2/+ and DTS1/+ animals showed a highly-localized region in the ventral lateral region of the central brain with increased pathology resulting in large holes in these regions.
Neurodegeneration was also observed in H64 and ATPalpha2206, which are both recessive loss-of-function ATPalpha alleles. In contrast to that seen in the dominant ATPalpha alleles, the neurodegeneration was less severe, especially in ATPalpha2206, and appeared as sporadically localized vacuolar pathology throughout the brain. In contrast to the massive degeneration seen in DTS1 and DTS2, seiTS2, another conditional mutant with a profound bursting physiological defect, showed only sporadic large vacuolar structures that were uncommon in age-matched control animals. Histological examination of each genotype (n>50, each genotype) demonstrated that the penetrance of the neuropathology observed in DTS1 and DTS2 was close to 100% and the distinctive patterns of neurodegeneration were reproducible for each mutant.
As is the case with many human neurodegenerative conditions, neurodegeneration in these mutant animals appears to be age-dependent and not the result of developmental defects. Young individuals of the ATPalpha mutants revealed little or no evidence of neurodegeneration in the brain or in sagittal sections of the thoracic ganglion, which were also examined for pathology in young and aged animals. In accord with the results found in the brain, the thoracic CNS also undergoes age-dependent neurodegeneration in DTS1 and DTS2 mutants.
To refine the cytological location of the mutants, we took advantage of the fact that DTS1 and DTS2 behave as dominant gain-of-function mutations and therefore should be revertible by second-site mutations within the gene that completely eliminate its function. Therefore, we screened for gamma ray-induced revertants of DTS1 and DTS2 that were not paralyzed at the restrictive temperature. Two revertants of DTS1 (DTSR1-R2) and three revertants of DTS2 (DTS2R1-R3) were recovered. DTS1R2, DTS2R1, and DTS2R2 were all associated with cytologically visible breakpoints in the 93A5-93B1,2 interval that disrupted ATPalpha and would be expected to abolish its activity.
The remaining two revertants, DTS1R1 and DTS2R3, were not associated with gross physical disruption of the ATPalpha gene by cytological or PCR analysis. However, direct sequence analysis of genomic DNA revealed that DTS1R1 is associated with a 4 bp deletion (ATPalpha deletion 2713-16) that results in a frameshift mutation in the ATPalpha coding region that is predicted to cause premature truncation of the protein product resulting in ATPalpha deletion 905-C, if any protein is actually made. Sequence analysis of DTS2R3 revealed two point mutations resulting in a predicted ATPalpha protein having E to A (39) and L to F (346) substitutions. Accordingly, all five DTS1 and DTS2 revertants have molecular defects in the ATPalpha gene consistent with loss-of-function mutations that can revert dominant TS mutations.
Each revertant contained a lesion in ATPalpha, confirming that the original DTS1 and DTS2 mutations, and by inference H64, are ATPalpha alleles. Each revertant contains mutations consistent with loss-of-function mutations and all but DTS2R2 appear to be null alleles of ATPalpha. We found no significant phenotypic differences among the five revertants and H64, suggesting that the H64 mutation may be a loss-of-function allele and that phenotypes arose in H64 and the revertants due to haploinsufficiency of ATPalpha. These data are consistent with the observation that a large deficiency, Df(3R)r-1G6/TM3, that removes ATPalpha causes a bang-sensitive phenotype (LEBOVITZ, 1989).
Molecular characterization of the revertants provided a strong indication that the original dominant mutations also resided in ATPalpha. We identified the original lesions associated with these mutants by direct sequence analysis of genomic DNA. Both ATPalphaDTS1 and ATPalphaDTS2 mutations cause predicted single amino acid substitutions in the C-terminus of the protein. This analysis revealed a single basepair change (G to A) in DTS2 which is predicted to cause a D to N substitution at position 981. DTS1 is also associated with a single basepair change (G to A) that results in an apparent E to K substitution of the next residue, at position 982. Both of these residues are conserved in ATPalpha proteins from representative species throughout the animal kingdom: Hs (Homo sapiens), Rn (Rattus norvegicus), Gg (Gallus gallus), Dr (Danio rerio), Ee (Electrophorus electricus), Dm (Drosophila melanogaster), Cf (Ctenocephalides felis, cat flea), As (Artemia franciscana, brine shrimp), Ce (Caenorhabditis elegans), Hv (Hydra vulgaris, hydra). The skilled artisan understands the degenerate three nucleotide codon system for encoding amino acids, and, being put in possession of the amino acid sequence of these mutations, is fully able to deduce numerous polynucleotides capable of encoding the mutant ATPalpha proteins of the invention.
It is extremely unlikely that these changes simply represent silent polymorphisms because they fall within a segment of the protein that is very highly conserved overall and the affected residues in particular are completely invariant in Na+/K+ ATPase alpha subunit proteins from hydra to human. In fact, these amino acid residues are even conserved in the more distantly related H+/K+ ATPase alpha subunit proteins. As expected, these substitutions are still present in the corresponding revertants, but are not found in any other control chromosome that we sequenced. It is striking that different alleles of the same gene, particularly two mutations affecting adjacent amino acids, could have such similarities in the patterns of neurodegeneration with different times of onset. These mutations may therefore identify key residues that serve important regulatory roles.
Prior proteolysis and chemical cross-linking experiments on Na+/K+ ATPase proteins have demonstrated that the C-terminus (M8-M10 region) of this protein makes intrasubunit contacts with the M1-M2 region as well as intersubunit contacts with the beta subunit. Without intending to be limited to a scientific theory underlying the invention, the dominant ATPalpha mutations of the invention could perturb one or both of these interactions, affecting regulation of the protein and resulting in gain-of-function phenotypes. Scanning mutagenesis of oxygen-containing residues predicted to be cytosolic or at the membrane/cytosol interface has been performed (Arguello (1999). As such, one of the residues in which we identified a lesion, D#981 (D995 in sheep Na+/K+ ATPase alphal isoform), has already been the subject of investigation. Those investigations demonstrate that the D995A mutations do not affect cation-enzyme interaction but do appear to impair protein maturation. The dominant phenotypes we observed in ATPalphaDTS1 and ATPalphaDTS2, which are more severe than those caused by null mutations of the same gene, suggest that these mutations cause a gain-of-function or have a dominant negative effect. Until recently it was thought that an alpha-beta protomer, the minimal unit of the enzyme required for function in vitro, was also the in vivo functional unit, making it more difficult to account for a dominant-negative effect. However, more recent data indicate that the protein may exist as a tetramer in vivo (Taniguchi, 2001). If the presence of even one mutant subunit could affect the activity or processing of the oligomeric complex, a dominant-negative effect could be readily explained.
The effect of these mutations was further verified by generating RT-PCR products from the ATPalpha mRNA isolated from DTS1, DTS2, H64 mutants and all of the revertants and then digesting these products with appropriate restriction enzymes to distinguish whether the RT-PCR products were derived from RNA transcribed off of mutant or wild-type alleles. In each case, the data demonstrated that the mutant chromosomes bearing the primary DTS1, DTS2 and H64 mutations still produced ATPalpha transcripts; however, there was no detectable expression of an ATPalpha transcript from the homolog containing DTS1R2, DTS2R1 and DTS2R2.
Without intending to be limited to any particular scientific theory underlying this aspect of the invention, the specific mutant residues identified in ATPalphaDTS1 and ATPalphaDTS2 may further destabilize ATPalpha protein resulting in some thermal unfolding at temperatures that are permissive for wild type ATPalpha. Scanning calorimetry studies of wild type pig kidney Na+/K+ ATPases have uncovered three domains of thermal unfolding, one mapping to the beta subunit and two to the alpha subunit (GRINBERG, 2001).
A profound temperature-dependent neuronal bursting electrophysiological phenotype was also noted in the DTS1 and DTS2 dominant mutants. Extracellular thoracic recordings (ETRs) were recorded essentially as described for electroretinograms, supra. Unevoked depolarization events (also referred to herein as hyperexcitability and bursting physiology) were seen predominately at elevated temperatures in ATPalpha and seizure mutant animals but do not appear in wild type. The bursting activity was apparently recorded extracellularly by the electrode positioned in the thorax just beneath the flight muscles and likely represents electrical activity in the dorsal longitudinal flight muscles in response to neuronal hyperexcitability in the flight motor pathway. This interpretation is supported by the observation of a similar, but somewhat more severe phenotype, for seits2, a K+ channel mutation known to cause neuronal hyperexcitability (Elkins, 1990; Titus, 1997). The bursting activity seen in DTS2 and DTS1 is consistent with defects in the sodium pump, which could result in more depolarized membrane resting potentials.
The physiological defect is present in very young adults, prior to the occurrence of any overt neurodegeneration. This result supports the conclusion that neural dysfunction, manifest as hyperexcitability, might lead to neuropathology. However, seits2, a mutation in the gene encoding ERG-type K+ channels, which also causes extensive bursting activity, is not associated with the kind of extensive neurodegeneration seen in ATPase alpha subunit mutants. These data suggest that hyperexcitability alone is not sufficient to cause neurodegeneration.
The physiological bursting phenotypes such as those seen in seits2 and reported here for dominant ATPalpha mutants have been observed in other Drosophila behavioral mutants. Such mutants are being used to investigate the physiological basis for seizure disorders such as epilepsy (KUEBLER, 2001). Our results demonstrate that ATPalpha is another gene that can cause physiological seizures when mutated in particular ways. The dominant ATPalpha mutants in Drosophila should provide a very useful experimental model for investigating physiological seizures, neurodegeneration, and the connection between them.
Example 3
Exon Analysis
The genomic locus of ATPalpha contains previously undescribed exons. Using primers directed towards exons 4 and 9 for RT-PCR reactions, we identified four exons that appeared to be mutually exclusive and named them 6a, 6b (formerly exon 6, NCBI database), 6c (formerly 13), and 6d. To date, all Drosophila ATPalpha cDNAs examined contained the exon corresponding to 6b (LEBOVITZ, 1989; SUN, 1998; Sun, 2001). Here, each of 123 ATPalpha clones contained one and only one member from the set of exons 6a, 6b, 6c, and 6d, indicating that these exons are utilized as mutually exclusive alternative cassettes. All four alternative exons are identical in length (94 bp). All of the newly described exons, as well as those previously known, appear to have wild type coding potential with the exception of the mutations discovered to affect residues 981 and 982, as described elsewhere herein. The sequences of new genomic exons 6a–6d are presented in the Sequence Listing as SEQ ID NO:2, 4, 6, and 8, respectively. The predicted amino acids encoded by genomic exons 6a–6d are presented as SEQ ID NO:3, 5, 7 and 9, respectively.
To clarify the exon 6 region, standard RT reactions were performed with a gene-specific primer directed to exon 9 (TTAATAGTAGGTCTCCTGCTCC-OH, SEQ ID NO:10), M-MLV Reverse Transcriptase (Promega) and 10 micrograms whole RNA isolated from embryos or adults using a modified LiCl/Urea preparation (AUFFRAY, 1980). Standard PCR reactions were performed as follows with primers directed toward exons 4 (TCAACACCGACGACATCAACTTCC-OH; SEQ ID NO:11) and 9 (GGTTGCGGCGCAAGTAGAAACGACG-OH, SEQ ID NO:12): 94° C. denaturing (45 seconds); 57° C. annealing (45 seconds); and 72° C. extension (2 minute), for 40 cycles. Products were cloned using the TOPO T/A Cloning Kit and One Shot E. coli (Invitrogen). Mini-plasmid preparations of transformants were analyzed by restriction digestion to determine which exon 6 was present. DraIII/EcoRI (NEB) double digests were diagnostic for exon 6b, and BsmBI (NEB) and BstYI (NEB) were diagnostic for exon 6c and 6d, respectively. Clones assayed negative for 6b,c and d were sequenced to verify that they contained exon 6a.
The alternative exons 6 encode part of the M6 transmembrane segment and the entire M6-M7 intracellular domain of the ATPalpha protein. Evolutionary comparisons show that this region is highly-conserved between worms and humans and that all three alternative exons encode most of these conserved residues. However, the three isoforms exhibit intriguing variations as well. In comparison with Na+/K+ ATPase alpha proteins of other species, exon 6c is most similar to the corresponding region of the nematode protein encoded by eat-6, and to the sequence encoded by the vertebrate orthologs. Several residues that vary among vertebrate Na+/K+ ATPase alpha paralogs and between H+/K+ ATPase alpha sequences vary among exons 6a, b, c, and d. These data suggest that Drosophila generates functionally diverse and structurally diverse ATPalpha proteins through alternative exon 6 splicing. Because the Drosophila genome contains one known Na+/K+ ATPase structural gene and one predicted paralog with good sequence homology (CG17923) rather than four, as appear to exist in most vertebrates, alternative splicing may be a mechanism for generating diversity in Drosophila comparable to that made available in vertebrates by the existence of several genes.
The cassette encoded by exons 6 extends from I/V797 to R827 (corresponding with V814 to R845 of sheep Na+/K+ ATPase alpha subunit), which encompasses the entire predicted cytosolic loop between M6 and M7 and part of the M6 transmembrane domain. Previous data have demonstrated that the M5-M6 region functions in cation binding and ion transport. Specifically, in mammals S775, Y771, E779, and F786 have been implicated in K+ coordination, Na+ interactions, voltage-dependence, and ouabain binding, respectively. In addition, D804 and D808, located in M6, are reported to be cation-coordinating residues. ADP binding has been shown to protect the M6-M7 cytoplasmic loop against tryptic digestion (LUTSENKO, 1994). The segment including YTLTSNIPEI (SEQ ID NO:13) in the fifth transmembrane segment is especially important in determining ion selectivity of the pump (PEDERSON et al, 1998). Together, these and other data demonstrate the central importance of the M5-M6 region to ATPase alpha function. The existence of multiple, alternatively spliced versions of exon 6 in the Drosophila ATPalpha gene suggest that the sequence differences encoded by these alternative exons could have profound functional consequences on pump kinetics, ion selectivity, or regulatory properties. Previous functional studies of ATPalpha in Drosophila have all utilized cDNAs that contained the same exon 6 splice variant (exon 6b). The discovery of multiple exons that generate additional structural diversity for this important region of the protein may reveal previously unsuspected functional diversity as well.
Example 4
Alternative 5′ End Splicing
In addition to alternative splicing in exon 6, ATPalpha also exhibits extensive alternative splicing at its 5′ end. Semi-quantitative RT-PCR and analysis of many isolated clones suggests that transcripts initiating near exon 12 are more abundant and diverse than those generated from exon 0. Semi-quantitative RT-PCR was performed on adult whole RNA as published, with some minor modifications (Palladino et al., 2000b). Primers directed to exon 0 and 12 sequences were separately used in combination with a reverse primer directed toward exon 3. RP49 primers directed to a gene for ribosomal protein were added to the reaction as a control at cycle 5. Samples were taken every other cycle from 16 and 28 and resolved on an agarose gel stained with ethidum bromide.
Quantification of gel fluorescence was performed on cycle 20 products using NIH image software. The RT-PCR products were cloned, as above, and representatives of each size clone were directly sequenced to document the splicing events. Exon 12 products became evident approximately 4 PCR cycles before those from exon 0, suggesting these transcripts are approximately 12–16 fold more abundant in adults. Consistent with this interpretation, fluorescence quantification revealed the ratio of ATPalpha products to RP49 product was 5.6±1.1 and 0.4±0.07 for exon 12 and exon 0 products, respectively (Error is SEM). Also, whereas an exon 0-directed upstream primer produces only one visible RT-PCR product (334 bp), at least four distinct products are evident when the upstream primer is directed toward exon 12 sequences, the most abundant of which are 577 bp and 349 bp.
Analysis of isolated clones is consistent with these interpretations and identifies the most abundant products from exon 12, as well as rare splice products. These analyses have identified a new multi-exon, 14/15, and a potentially new translational start in exon 15. These data suggest that three alternate N-termini exist for this protein; two long forms with putative translational initiation sites in exon 0 and 15 (amino acid sequences are provided as SEQ ID NO:14 and SEQ ID NO:15, respectively), and a short form with an initiation site in constitutive exon 2 (amino acid sequence is provided as SEQ ID NO:1). The same neurodegeneration mutations on SEQ ID NO:14 that correspond to mutations at residues 981 and 982 on SEQ ID NO:1 are at residues 1020 and 1021 of SEQ ID NO:14, respectively; the same neurodegeneration mutations on SEQ ID NO:15 that correspond to mutations at residues 981 and 982 on SEQ ID NO:1 are at residues 1016 and 1017 of SEQ ID NO:14, respectively.
In summary, Examples 2, 3 and 4 demonstrate again that temperature-sensitive and bang-sensitive mutants are an advantageous pool from which to screen for neurodegeneration mutants. In these embodiments, the neurodegeneration mutants were further characterized at the molecular level. These data establish Drosophila as an important model that can be used to better understand important human disease conditions associated with the affected gene and its biochemical pathways, and, by extension, for other conditions associated with other genes identified in the selecting and screening method of the invention. The molecular analysis of ATPalpha promoter usage and alternative splicing complements the mutant analysis and helps to further develop this important model.
Example 5
We have described in previous examples Na/K ATPase alpha subunit (ATPalpha) alleles and vacu mutants that manifest a neurodegenerative phenotype. All of these alleles, and in fact any allele with a loss of function mutation in the ATPase alpha subunit, can be used for evaluating the neuroprotective agent. In addition to the ATPalpha alleles, we have similarly identified other neurodegenerative alleles that can be used for evaluating neuroprotective agents.
One of these alleles we identified, has a lesion in a gene known as CG4684, the sequence of which is provided as SEQ ID NO:16. The lesion results in a P866Q in an allele known as 565. The wild type (SEQ ID NO:16) and the lesion (P866Q) proteins are within the scope of the present invention. In addition, polynucleotides that encode the above proteins are also within the scope of the present invention. This allele can be used in the method of the present invention for evaluating neuroprotective agents.
Littleton et al, Proc Natl Acad Sci USA 88 (21): 12233–12238 (2001), which is herein incorporated by reference in its entirety, disclosed several NSF1 alleles: TP7 (P398S), ST53 (S483L), ST17 (G274E) and G4 (VQQ525-527GS). The amino acid sequence of the NSF1 protein can be found with NCBI accession number P46461 (provided as SEQ ID NO:17). We have found that these alleles manifest a neurodegenerative phenotype and thus can be used in the method of the present invention for evaluating neuroprotective agents.
Mol Gen Genet 256 (6): 602–610 (1997), which is herein incorporated by reference in its entirety, disclosed alleles with lesions on a voltage-dependent sodium channel (paralytic or para) with two lesions specifically identified: TS1 (1265N), DTS2 (A1506V), DTS3, ST109, TS 115. The amino acid sequence of the protein can be found with Swiss Protein accession number P35500 (provided as SEQ ID NO:18). We have found that these alleles manifest a neurodegenerative phenotype and thus can be used in the method of the present invention for evaluating neuroprotective agents.
Science 283 (5406): 1343–1345 (1999), which is herein incorporated by reference in its entirety, disclosed several axotactin alleles: 52, 8D and 6. They are all loss-of function alleles with non-specified disruptions in the axo gene. We have found that these alleles manifest a neurodegenerative phenotype. Thus these alleles and any other alleles that carry loss-of-function lesions on axotactin can be used in the method of the present invention for evaluating neuroprotective agents. Axotactin amino acid sequence is provided as SEQ ID NO:19. Partial axotactin amino acid sequence can be found with accession Fban0018296.
In J Biol Chem 274 (31): 22109–22113 (1999), which is herein incorporated by reference in its entirety, a Shab allele was disclosed: Shab1 (R435Q). The amino acid sequence of the protein can be found with Swiss Protein accession number P17970 (provided as SEQ ID NO:20). We have found that the allele manifests a neurodegenerative phenotype and thus can be used in the method of the present invention for evaluating neuroprotective agents.
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## Claims

1. A method for evaluating a putative neuroprotective agent, the method comprising the steps of:
administering a putative neuroprotective agent to a Drosophila strain susceptible to age-dependent neurodegeneration, the strain being a behavioral mutant selected from the group consisting of a temperature-sensitive paralytic mutant and a bang-sensitive paralytic mutant; and
evaluating a neuroprotective effect of the agent.

2. The method as claimed in claim 1 wherein a characteristic of the neuroprotective effect is selected from the group consisting of a reduction in severity of neurodegeneration, a delay in onset of neurodegeneration, an improved age-dependent behavior, and an increase in lifespan of the strain.

3. The method as claimed in claim 1 wherein the neuroprotective effect is evaluated by a histological screen.

4. The method as claimed in claim 1 wherein the agent is administered by a method selected from the group consisting of feeding the agent to the strain and injecting the agent into the strain.