Inositol 1,4,5-trisphosphate metabolism in the cerebella of Lurcher mutant mice and patients with olivopontocerebellar atrophy

Journal of the Neurological Sciences, 110 (1992) 139-143 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00

139

JNS 03790

Inositol 1,4,5-trisphosphate metabolism in the cerebella of Lurcher mutant mice and patients with olivopontocerebellar atrophy P.J.S. Vig, S.H. S u b r a m o n y , R . D . C u r r i e r a n d D. D e s a i a h Department of Neurology, Unit,ersityof MississippiMedical Center, Jackson, MS 39216, USA (Received 6 September, 1991) (Revised, received 31 January, 1992) (Accepted 8 February, 1992)

Key words: lnositoi 1,4,5-trisphosphate; Olivopontocerebeilar atrophy; Lurcher mouse

Summary We have investigated inositol 1,4,5-trisphosphate (InsP3) metabolism in cerebellar membranes of normal humans and patients with dominant ataxia ('C' kindred), and also in cerebellar microsomes of Lurcher mutant mouse (a suggested model for cerebellar ataxia). Various [aH]InsP3 metabolites formed were separated by HPLC using 3 successive convex gradients of 1.TM ammonium formate, pH 3.7. [3H]InsP3 metabolism was rapid and in 15- and 45-day-old control mice cerebella about 50% of [3H]InsP3 was metabolized within 20 s. In 15-day-old Lurcher mice the rate of [3H]InsP3 metabolism was significantly low (40% of normal). [3H]InsP3 metabolism was almost absent in 45-day-old Lurcher mice cerebellar microsomes. The decreased [3H]InsP3 metabolism was consistent with decreased recovery of the various inositol polyphosphates formed. Similarly, in cerebellar membranes of human patients with olivopontocerebellar atrophy (OPCA) a significant decrease in [aH]InsP3 metabolism was observed when compared with normal controls. These data suggest that altered phosphoinositide turnover may be associated with the onset of neuronal degeneration in human OPCA.

Introduction Olivopontocerebellar atrophy (OPCA), like other hereditary ataxias, is a neurological disorder that affects a significant number of people in the U.S. The disease is dominantly inherited and the children and siblings of the affected are at risk. OPCA is characterized clinically by cerebellar ataxia and pathologically by degeneration of Purkinje and granule cells in the cerebellum, inferior olivary neurons and pontine nuclei. There is no available treatment for this disorder (Bebin et al. 1990). Biochemical studies have shown decreased levels of aspartate, glutamate, y-aminobutyric acid and an increase in taurine (Perry et al. 1977, 1981; Perry 1984). These studies have contributed to some extent to the understanding of the underlying biochemical abnormalities in these disorders. However, no relationship between the changes of these neurochemicals and the degenerative process has been established. Lurcher mutant mouse (gene symbol: LC) is an

Correspondence to: D. Desaiah, Professor, Department of Neurology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. Tel.: (601) 984-5511; Fax: (601) 984-5503.

autosomal dominant mutation that resembles adult dominant ataxia of the OPCA type in its clinical, genetic and histological features (Phillips 1960; Caddy and Biscoe 1975). The Lurcher heterozygotes can be easily recognized at 13-15 days of postnatal age, by their lurching movement and by their light coat color (Caddy and Biscoe 1975). Pathologically these animals reveal loss of Purkinje cells as well as involvement of granule and inferior olivary neurons (Caddy and Biscoe 1979). Thus, the Lurcher mutant is an appropriate animal model for cerebellar ataxias of the OPCA type. In recent years it has been shown that a variety of compounds such as neurotransmitters, neuropeptides, hormones and growth factors produce their diverse physiological responses by interaction with specific cell surface receptors (Berridge and Irvine 1984; Chuang 1989). The stimulation of these receptors triggers the production of second messengers. One such pathway, critical to an array of physiological processes in the central nervous system, is the phosphoinositide (Pl)-derived dual second messenger system (Berridge and Irvine i984; Hir~sawa and Nishizuka 1985; Berridge 1987). Receptor activation is known to increase the hydrolysis of Pls to yield inositol 1,4,5-trisphosphate (InsP 3) and diacylglycerol (DG) (Berridge 1984). InsP 3 regulates the release of Ca 2+ from intracellular or-

140 ganelles such as endoplasmic reticulum whereas DG is shown to activate protein kinase C (PKC) (Williamson et al. 1985; Berridge 1987). Both Ca 2+ and PKC are known regulators of synaptic transmission, membrane excitability, cellular metabolism, growth and DNA synthesis (Zurgil and Zispel 1985; Shuntoh and Tanaka 1986; Berridge 1987; Kaczmarek 1987). Any fluctuation in these intracellular regulators can cause severe cellular dysfunction. Recently, it has been reported that degenerating process in human OPCA and in Lurcher mutant mouse cerebellum is associated with a significant decrease in InsP3-receptor binding (Kish et al. 1989; Desaiah et al. 1991) and InsP3-mediated cae+-release from cerebellar microsomes (Desaiah et al. 1991). There was also a significant reduction of PKC activity in the cerebellar cytosolic fractions. The decrease was specific to cerebellum as there was no change in cerebral cortex (Desaiah et al. 1991). These biochemical alterations were observed in 15-day-old mice, the day of onset of ataxia (Caddy and Biscoe 1975, 1979). The present study was initiated to test the hypothesis that the , progression of cerebellar degeneration is associated with altered InsP3 metabolism that triggers the degenerative process le~,ding to neuronal death.

Lurcher male) per cage. Water and pelleted Purina Chow were provided ad libitum. Cerebella of 15-, 25-, and 45-day-old mice were removed and quick-frozen in liquid nitrogen. The unaffected littermates were used as controls.

Mouse cerebellar microsomes

Materials and method~

The cerebellar microsomes were prepared by the method of Stauderman et al. (1988) as described previously (Desaiah et al. 1991). Briefly, cerebellar tissue was homogenized in 9 vols. of microsome-preparation buffer (MPB) containing 250 mM sucrose, 5 mM Hepes (pH 7.05), 10 mM KCi, 1 mM dithiothreitol and 1 mM MgCl 2. The homogenate was centrifuged at 1000 × g for 5 min and the resulting supernatant fraction (S~) was kept on ice while the pellet was washed once in 10 ml of MPB. The S~ supernatant fractions were combined and then centrifuged at 8000 × g for 10 rain, after which the supernatant fraction ($2) was kept and the pellet was washed once in 10 ml of MPB. The S 2 supernatant fractions were combined and centrifuged at 100000 × g for 60 min to obtain the microsomal pellet (P3) which was used in all experiments. The microsomal pellet was resuspended in a buffer containing 150 mM KCI, 5 mM Hepes (pH 7.05) and 2 mM MgCI 2. The suspension in aliquots of 0.5 ml was frozen in liquid nitrogen and stored at -80°C till further use.

Human patients

Human cerebellar membranes

Autopsied cerebellar tissues from 4 normal controls who died of non-neurological cause and 4 patients with OPCA from one family (the 'C' kindred) were used (Currier et al. 1972; Desaiah et al. 1991). The mean age of control subjects was 29 years and OPCA patients was 46 years at the time of death. The mean postmortem time in controls was 14 h and in OPCA patients was 6 h. The OPCA patients were diagnosed based on family history, clinical signs and characteristic histological changes in the cerebellum (Currier et al. 1972). The OPCA patients suffered from imbalance, ataxia of limbs and dysarthria. Neuropathological studies revealed severe atrophy of inferior olives, pons and cerebellum. Histological analysis in the OPCA patients showed Purkinje and granule cell loss in cerebellar cortex. The density of neurons in the inferior olive was also decreased in all of these patients. All the OPCA patients were severely disabled by their end-state cerebellar ataxia and were wheelchair- or bed-bound The duration of ataxia was 15-30 years.

The cerebellar membranes were prepared according to the method of Theibert et al. (1987). Briefly, cerebellar tissue was homogenized in 50 vols. of 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, pelleted by centrifugation at 35 000 × g for 10 min, washed twice in homogenizing buffer and finally resuspended in appropriate volume of 150 mM KCl, 5 mM Hepes (pH 7.05), 2 mM MgC! 2. The resuspended pellet was frozen in liquid nitrogen and stored at - 8 0 ° C till further use. Cerebellar membrane proteins were estimated according to the method of Lowry et al. (1951).

Lurcher mice Breeding pairs of Lurcher ( L C / + ) mice were obtained from the Jackson Laboratories (Bar Harbor, ME). Animals were housed in clean polypropylene cages with one breeding pair (normal female and

Metabolism of l SHIlnsPs The procedure of Stauderman et al. (1988) was used to study the [3H]InsP3 metabolism. The reaction mixture (final volume 1 ml) contained 100 nM [SH]InsP3 (specific activity 17.0 Ci/mmol), 150 mM KCI, 5 mM Hepes (pH 7.05), 2 mM MgCI2, 0.5 mM ATP, 10 mM phosphocreatine, 10 units/ml creatine kinase, 1/~g/ml oligomycin and 1 mM NaN 3 and about 500 /~g of microsomal or cerebellar membrane protein. The microfuge tubes containing the reaction mixture were preincubated for 5 min at 37°C. The reaction was initiated by the addition of microsomes or cerebeilar membranes. After 20 s, 1 ml ice-cold 20% (w/v) trichloroacetic acid (TCA) was added to the reaction

141 mixture. For the control (zero time) the microsomal or membrane preparations were quenched with TCA before the addition of [3H]lnsP3. After quenching the samples were kept on ice for 10-15 min, with intermittent vortex-mixing, and then centrifuged at 10000 x g for 5 min. The resulting supernatant fraction was extracted with 4 x 4 vols. of water-saturated diethyl ether and then neutralized with NH4OH. Separation of the various [3H]inositol phosphates was performed by HPLC, with a Whatman Partisil SAX column (10/tm particle size). The [3H]inositol phosphates were eluted by 3 successive convex gradients of ammonium formate (pH 3.7). The concentration steps of 0.5 M, 1.0 M and 1.7 M, each over 15-min intervals were employed. The gradient was generated by Waters 510 pumps connected to a System Interface Module, controlled by a Digital 380 computer. The flow rate was 1.2 ml/min and fractions were collected every 60 s. To each fraction 10 mi of 'Biofluor' high-efficiency emulsifier cocktail (NEN/Dupont) was added and the radioactivity was determined by a liquid scintillation counter. Identification of HPLC peaks containing [3H]Ins(1,4)P2, [3H]Ins(1,3,4)1)3, [ 3H]lns(1,4,5)P3 and [3H]lns(1,3,4,5)1)4 was made by comparison with standards.

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Fig. 1. Elution profile of inositol phosphates by HPLC. The samples analyzed were a neutralized trichloroacetic acid extract of mouse cerebellar microsomes. The metabolites of [3H]Ins(l,4,5)Pa were separated by HPLC as described in Materials and Methods.

Ins(1,4)P2 !

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Statistical analysis Data are expressed as the mean + SEM. From normal and ataxic human cerebella, 300-500 mg tissue was taken and was processed individually. Lurcher mice data represent a minimum of 3 different preparations assayed in duplicate. Each assay was done by pooling 6 cerebella. Statistical significance was calculated by using the two-sample t-test for independent samples. A value of P < 0.05 was accepted as statistically significant.

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Fig. 2. Analysis of inositol phosphates by HPI.(2. The samples analyzed were a neutralized trichloroacetic acid extract of human cerebellar membranes. The various inositol phosphates formed due to the metabolism of [3H]lns(l,4,5)P3 were separated by HPLC as described in Materials and Methods.

Results

Elution profiles of mouse cerebellar microsomes (Fig. 1) and human cerebellar membrane ( F i g . 2) fractions showed gradual appearance of various [3H]inositol phosphates formed as a result of [3H]InsP3 metabolism. The peaks were identified by comparing with the elution of the standard [3H]inositol phosphates. The various inositol phosphates were recovered with the increasing ammonium formate concentration. Ins(1,4)P2 appeared first followed by Ins(1,3,4)P3, lns(1,4,5)P3 and Ins(1,3,4,5)P4. Although other metabolites of [3H]Ins(1,4,5)P3 were also eluted, the peaks were near the detection limit of HPLC and have not been presented in these data. The analysis of the data revealed that there was a significant increase in the recovery of [3H]InsP3 in the 15 days Lurcher mouse cerebellum when compared with the littermate controls (Fig. 3) suggesting a de-

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Ins(1,S,4)P3

Ins(1,4,5)P3 Ins(1,S,4,5)P4

Fig. 3. Decreased metabolism of InsP 3 in mouse cerebellar microsomes. Microsomes were prepared from cerebella of 15-day-old Lurcher mice and their iittermate controls. The metabolism of [3H]insP3 (100 nM) was studied by incubating the microsomal preparations with [3H]InsP3 for 20 s and separating the newly formed metabolities by HPLC as described in Materials and Methods. * P < 0.05.

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Fig. 4. Inhibition of InsP3 metabolism in mouse cerebellar microsomes. Cerebellar microsomes were prepared from 45-day-old Lurcher mice and their littermate controls. [3H]lnsP3 metabolism was studied as described in Materials and Methods. * P < 0.05.

crease in InsP3 metabolism in Lurcher mouse microsomes. Since, [aH]InsP3 metabolism was inhibited, the recovery of [aH]Ins(1,4)P2, [3H]Ins(1,3,4)P3 and [3H]Ins(1,3,4,5)P4 also decreased significantly (Fig. 3). Similar data were obtained for 45 days Lurcher mouse cerebellum. However, the [3H]InsP3 metabolism was almost completely inhibited (Fig. 4). Crude human cerebellar membrane preparations also exhibited the identical data as that of Lurcher mouse cerebellar microsomes (Fig. 5). The [3H]Ins(1,4,5)P3 recovery in the cerebellum of OPCA patients increased significantly when compared with normal controls, whereas [aH]Ins(1,4)P2 formation was signficantly inhibited. [3H]Ins(1,3,4,5)P4 was not detected in crude human cerebellar membrane fractions except one sample, which also showed very low recovery of [3H]Ins(1, 3,4,5)P4.

Discussion The results of the present study clearly show that the [3H]lnsP3 metabolism is decreased in Lurcher mouse and in human OPCA cerebellum. The 15-dayold Lurcher mouse data suggest the possible biochemical changes at or before the onset of ataxia. On the 15th day, in Lurcher mouse cerebellum, the Purkinje cell loss is minimal (Caddy and Biscoe 1975). However, the cells have irregular cell and nuclear membrane and disordered endoplasmic reticulum (Caddy and Biscoe 1975). The observed biochemical alterations may be related to the impaired functional capacity of endoplasmic reticulum. These observations are supported by our previous findings (Desaiah et al. 1991) where we have demonstrated the inhibition of InsP3 receptor binding and InsP3-mediated Ca2÷-release from human OPCA patients and Lurcher mouse cerebellar microsomes. Ins(l,4,5)P 3 is predominantly metabolized to lns(1,4)P2 and Ins(l,3,4,5)P4 in membrane fractions by

the enzymes 5-phosphatase and 3-kinase, respectively. The particulate 5-phosphatase is associated with plasma membrane (Shears 1989). Since Ins(1,4)P 2 does not mobilize Ca 2+ (Berridge 1987), 5-phosphatase plays a vital role in terminating the release of C a 2+ b y Ins(1,4,5)P3. 3-Kinase on the other hand is a soluble enzyme but is also shown to be present in a significant amount in the membranes (Stauderman et al. 1988; Shears 1989). 3-Kinase also plays a key role in regulating lnsP3 metabolism. Since 3-kinase is a Ca 2+calmodulin-dependent enzyme (Biden and Wollheim 1986; Morris et al. 1987; Rossier et al. 1987; Biden et al. 1988), the transient receptor-mediated elevation of cytosolic Ca 2+ can increase the rate of formation of Ins(1,3,4,5)P4. This phenomenon may enhance the Ca2+-mobilizing function of Ins(1,3,4,5)P 4 and thus inhibit Ins(1,4,5)P 3 dephosphorylation (Irvine and Moor 1986; Parker and Miledi 1987). The decreased InsP3 metabolism, due to the decreased activities of 5-phosphatase and 3-kinase, can lead to excessive accumulation of Ins(1,4,5)P 3 within the cell. Furthermore, uncontrolled Ca2+-release may alter Ca 2+ homeostatic mechanisms leading to neuronal injury. Blackstone et al. (1989) have shown the absence of glutamate-enhanced PI turnover in Purkinje-cell-deficient mice. However, this observation has been attributed to the complete loss of Purkinje cells (> 99%) as in the littermate controls glutamate produced a significant increase in PI turnover. In ataxic cerebellum we have observed decreased PKC and lnsP3 receptor activity (Desaiah et al. 1991), and the data from the present study suggest that in OPCA the key regulatory steps of phosphoinositide-signalling pathway are impaired. Since a significant decrease in InsP3 metabolism was evident on the 15th day, the day of onset of ataxia in Lurcher mouse cerebellum, it can be speculated that the altered phosphoinositide turnover and/or Ca 2+homeostasis may play a key role in the degenerative

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Fig. 5. Inositol 1,4,5-trisphosphate metabolism in human cerebellar membranes. The cerebellar membranes from the normal controls (n = 4) and OPCA patients (n -- 4) were prepared as described in the Materials and Methods. The cerebellar membranes were incubated (for 20 s) with 100 nM [3H]lnsP3. The various metabolites formed were separated by HPLC. * P < 0.05.

143

process. Alternatively, this may be a nonspecific manifestation of cytosolic dysfunction triggered by the primary genetic defect. However, studies on cultured Lurcher mouse cerebellar neurons may provide a better insight to the mechanism of neuronal degeneration and the role of Ca 2+ in cell death. Acknowledgement This work was supported by the National Ataxia Foundation, Minneapolis, MN, U.S.A

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