The tocopherol transfer protein mediates vitamin E trafficking between cerebellar astrocytes and neurons

Abstract

Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein–positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor–related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress–related neurodegenerative diseases. Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein–positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor–related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress–related neurodegenerative diseases. Vitamin E is a collective term that denotes a family of eight neutral plant lipids (1Ulatowski L. Manor D. Vitamin E trafficking in neurologic health and disease.Annu. Rev. Nutr. 2013; 33: 87-103Google Scholar), of which α-tocopherol is selectively retained in the body and considered the most biologically active form of the vitamin (2Burton G.W. Cheeseman K.H. Doba T. Ingold K.U. Slater T.F. Vitamin E as an antioxidant in vitro and in vivo.Ciba Found. Symp. 1983; 101: 4-18Google Scholar, 3Ingold K.U. Webb A.C. Witter D. Burton G.W. Metcalfe T.A. Muller D.P. Vitamin E remains the major lipid-soluble, chain-breaking antioxidant in human plasma even in individuals suffering severe vitamin E deficiency.Arch. Biochem. Biophys. 1987; 259: 224-225Google Scholar). The potent radical-trapping activity of α-tocopherol is thought to prevent free radical–induced lipid peroxidation, lending the vitamin its distinction as the major lipid-soluble antioxidant in most species. Consequently, adequate vitamin E status is considered an essential line of physiological defense against oxidative stress–related diseases. The essential requirement for α-tocopherol for maintaining central nervous system (CNS) health has been recognized for over 50 years. Secondary vitamin E deficiency accompanying fat malabsorption diseases, such as abetalipoproteinemia, cholestasis, and short bowel syndrome, manifests in CNS dysfunctions that can often be remedied by timely supplementation with α-tocopherol (4Muller D.P. Lloyd J.K. Effect of large oral doses of vitamin E on the neurological sequelae of patients with abetalipoproteinemia.Ann. N. Y. Acad. Sci. 1982; 393: 133-144Google Scholar, 5Runge P. Muller D.P. McAllister J. Calver D. Lloyd J.K. Taylor D. Oral vitamin E supplements can prevent the retinopathy of abetalipoproteinaemia.Br. J. Ophthalmol. 1986; 70: 166-173Google Scholar, 6Sokol R.J. Butler-Simon N. Conner C. Heubi J.E. Sinatra F.R. Suchy F.J. Heyman M.B. Perrault J. Rothbaum R.J. Levy J. Multicenter trial of d-alpha-tocopheryl polyethylene glycol 1000 succinate for treatment of vitamin E deficiency in children with chronic cholestasis.Gastroenterology. 1993; 104: 1727-1735Google Scholar, 7Sokol R.J. Heubi J.E. Butler-Simon N. McClung H.J. Lilly J.R. Silverman A. Treatment of vitamin E deficiency during chronic childhood cholestasis with oral d-alpha-tocopheryl polyethylene glycol-1000 succinate.Gastroenterology. 1987; 93: 975-985Google Scholar, 8Sokol R.J. Guggenheim M.A. Iannaccone S.T. Barkhaus P.E. Miller C. Silverman A. Balistreri W.F. Heubi J.E. Improved neurologic function after long-term correction of vitamin E deficiency in children with chronic cholestasis.N. Engl. J. Med. 1985; 313: 1580-1586Google Scholar, 9Traber M.G. Schiano T.D. Steephen A.C. Kayden H.J. Shike M. Efficacy of water-soluble vitamin E in the treatment of vitamin E malabsorption in short-bowel syndrome.Am. J. Clin. Nutr. 1994; 59: 1270-1274Google Scholar). The unique metabolic profile of the CNS, coupled with high levels of polyunsaturated fatty acids in neurons and astrocytes, renders this tissue particularly vulnerable to lipid peroxidation and subsequent cellular damage. Indeed, elevated levels of oxidative stress markers accompany a variety of neuropathological conditions. Analyses of autopsy samples from human patients afflicted with Alzheimer's disease (10Lovell M.A. Ehmann W.D. Butler S.M. Markesbery W.R. Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer's disease.Neurology. 1995; 45: 1594-1601Google Scholar, 11Montine T.J. Markesbery W.R. Morrow J.D. Roberts 2nd, L.J. Cerebrospinal levels are in Alzheimer's Scholar), disease F.R. Y. A. P. lipid peroxidation in is in Scholar), and Markesbery W.R. by the lipid peroxidation in the of elevated levels of oxidative stress lipid peroxidation α-tocopherol in plasma and from Alzheimer's disease and patients are in individuals C. M.B. peroxidation and free in Alzheimer's Scholar, A. M. and oxidative stress markers in and plasma from Scholar, M. L. T. of in patients with of the Nutr. Scholar, T. M. of alpha-tocopherol and its in from patients with of the and 1994; Scholar, F.J. J. A. T. M. J. Cerebrospinal levels of alpha-tocopherol (vitamin E) in Alzheimer's 104: Scholar, M. A. of vitamin vitamin and and the of Scholar). supplementation with vitamin E to the levels of oxidative stress markers and to disease in human patients and in of Alzheimer's and M. A. of vitamin vitamin and and the of Scholar, Vitamin and implications for neurodegeneration in N. Y. Acad. Sci. Scholar, C. D. C. of treatment in large disease trial Scholar, Vitamin E in Scholar, C. in Alzheimer's disease as and Med. 33: Scholar, C. in Alzheimer's disease and as Scholar, M. C. M. P. J. L.J. trial of as treatment for Alzheimer's The Alzheimer's Engl. J. Med. Scholar, M. of vitamin E on the of disease in and Scholar, M. Vitamin E and The for J. Clin. Nutr. Scholar, M.G. A. A. Vitamin E and of of data from J. Scholar). These the for α-tocopherol supplements to patients afflicted with C. in Alzheimer's disease as and Med. 33: Scholar, C. in Alzheimer's disease and as Scholar, Vitamin E supplementation in Alzheimer's and Scholar). of results from of and of Scholar, C. M. C. A. trial of alpha-tocopherol (vitamin E) in the treatment of Scholar), the of vitamin E as a in neurodegenerative remains A. J. that lipid peroxidation and its sequelae in Alzheimer's disease brain to Scholar). the the alpha-tocopherol transfer protein (TTP) facilitates the secretion of α-tocopherol from to for delivery to whole-body status of the vitamin M. Alpha-tocopherol transfer protein the secretion of alpha-tocopherol from a cultured line a Acad. Sci. A. Scholar, J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar, D. The alpha-tocopherol transfer Scholar). The role of TTP is by the that mutations in the TTPA gene the familial with vitamin E deficiency in L. Kayden H.J. L. M. with vitamin E of mutations and in a large of J. Scholar, T. M. T. Y. Y. N. spinocerebellar by a in the gene for the Engl. J. Med. 1995; Scholar, M. Kayden M. M. with vitamin E deficiency is by mutations in the alpha-tocopherol transfer 1995; is by plasma elevated levels of oxidative stress and as progressive spinocerebellar and M. Kayden M. M. with vitamin E deficiency is by mutations in the alpha-tocopherol transfer 1995; Scholar, and molecular of cerebellar Scholar, R.J. Vitamin E and neurologic Scholar). the that the of TTP mutations are and TTP is expressed in CNS A. T. T. M. M. J. of alpha-tocopherol transfer protein in Scholar, T. A. M. Kayden H.J. of alpha-tocopherol transfer protein in the of patients with with vitamin E deficiency and oxidative stress neurodegenerative Scholar), is the and mechanisms of of TTP in the We our on the of TTP in cerebellar astrocytes and its role in maintaining vitamin E in neurons as as of oxidative stress. The of TTP in transport have been primarily in its of the D. The alpha-tocopherol transfer Scholar, M.G. mechanisms of vitamin E Rev. Nutr. Scholar). the protein is also expressed in the and A. T. T. M. M. J. of alpha-tocopherol transfer protein in Scholar, T. A. M. Kayden H.J. of alpha-tocopherol transfer protein in the of patients with with vitamin E deficiency and oxidative stress neurodegenerative Scholar, M. J. alpha-tocopherol transfer Scholar, J. Alpha-tocopherol transfer protein is the of Scholar), where its functions are poorly understood. results in neurological L. Kayden H.J. L. M. with vitamin E of mutations and in a large of J. Scholar, M. Kayden M. M. with vitamin E deficiency is by mutations in the alpha-tocopherol transfer 1995; Scholar), we our to the CNS, the we found that the murine TtpA is expressed in of the and that this with of TtpA in the is in is in the and is with of the TtpA in the is and is brain in the A. T. T. M. M. J. of alpha-tocopherol transfer protein in the profile of the TTP in the brain that of the TTP protein These data and our results L. C. N. J. Manor D. of the α-tocopherol transfer protein gene is by oxidative stress and Med. Scholar), that of TTP is and The high of TTP in the and our of of cerebellar neurons in L. Manor D. Vitamin E is essential for with the cerebellar by patients L. Kayden H.J. L. M. with vitamin E of mutations and in a large of J. Scholar, M. Kayden M. M. with vitamin E deficiency is by mutations in the alpha-tocopherol transfer 1995; Scholar, M. M. M. A. C. Treatment of in vitamin E deficiency by alpha-tocopherol transfer protein address the function of TTP in the cerebellum, we first the distribution of the protein in the murine We in from in from of the TTP protein in the to the neurons, most A. T. T. M. M. J. of alpha-tocopherol transfer protein in The CNS is of neurons, and in can be into astrocytes and on the in which TTP is we cultured and that selectively We with against an fibrillary acidic protein C. M. M. J. of glial fibrillary acidic protein in human brain Sci. Scholar, L.J. A. M.A. and of human glial fibrillary acidic Acad. Sci. A. a A. L.J. of and in the of a murine for and J. Scholar, L.J. of the and of human Scholar, M.A. distribution of in J. that the that in neurons astrocytes and We which of these that is expressed levels in astrocytes as with These are by a profile in which to be high in murine astrocytes in neurons J.D. A. Y. for astrocytes, neurons, and for understanding brain and Scholar). We the of the TTP protein in cultured where are we found that TTP is expressed in astrocytes, where it is the and We TTP in neurons we the of TTP in where the cellular tissue and of are M. D. S.M. has of Scholar, D. P. of the and Scholar). we of TTP in in neurons and of the a the of TTP and of TTP in Taken together, our data that TTP and protein is selectively expressed in astrocytes and in is expressed in astrocytes. and cerebellar and cerebellar and from an in to with against TTP and and and the and the and the in the of and the The glial fibrillary acidic alpha-tocopherol transfer We the where α-tocopherol in the the uptake and distribution of the we a of vitamin E that we have to be a of vitamin E in vitro and in J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar, M. P. J. Manor D. of a vitamin E as a for transfer protein 45: Scholar, P. M. Manor D. J. of for in protein and with the alpha-tocopherol transfer Med. Scholar, J. J. Manor D. of mutations in the alpha-tocopherol transfer 45: Scholar, D. J. J. Manor D. transfer protein to vitamin Scholar, M. A. N. J. Manor D. of transfer by the transfer Scholar). is thought to to lipoprotein J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar, M.G. mechanisms of vitamin E Rev. Nutr. Scholar, M.A. Muller D.P. and uptake of alpha-tocopherol and low-density lipoprotein by in and control J. Scholar, M.G. Kayden H.J. Vitamin E is to the high affinity for low-density J. Clin. Nutr. Scholar, M.G. Ingold K.U. Burton G.W. Kayden H.J. and transport of in human Scholar, vitro uptake of from lipoprotein by cultured human Nutr. 1985; Scholar, as a for the delivery of and alpha-tocopherol to Med. Scholar, M. A.C. is the major of vitamin E for Med. Scholar, D. A. is expressed in brain and to uptake of vitamin Scholar), we to M. Alpha-tocopherol transfer protein the secretion of alpha-tocopherol from a cultured line a Acad. Sci. A. Scholar, is the of alpha-tocopherol and transfer and J. Biochem. Scholar, A. of from the of J. Biochem. to E of vitamin E in and and the uptake of vitamin E by of Biophys. Scholar, E deficiency to brain uptake of into Biophys. with the cultured and by in astrocytes as with neurons, where in These observations that TTP-expressing astrocytes vitamin and that of TTP in these regulates the distribution in the The of vitamin E important into the mechanisms that its of vitamin E distribution in in J.D. are in 70: Scholar, The alpha-tocopherol and fatty acids in Biophys. and A. Effect of deficiency and supplementation with on in Nutr. Scholar), the of vitamin E of the brain has been the where vitamin E we cerebellar with and the of the vitamin as with observations indicate that the levels in astrocytes as with neurons in neurons and astrocytes from we the vitamin and the and that of vitamin E into astrocytes and neurons the also with the in astrocytes and in neurons the distribution of in cerebellar from in in astrocytes, where it with and to a with and These observations show that the of TTP uptake of vitamin TTP vitamin E trafficking by secretion of α-tocopherol from the as it in J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar). this we with and secretion of the vitamin into the over a J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar). We found that astrocytes vitamin E to a neurons the of vitamin E secretion from astrocytes to that in TTP-expressing where the physiological function of TTP is J. P. J. Manor D. trafficking of vitamin E in The role of transfer these we the of on the levels of the vitamin. We astrocytes with and its over after from by we have this in secretion of the vitamin from the astrocytes to the J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar, P. M. Manor D. J. of for in protein and with the alpha-tocopherol transfer Med. Scholar, D. J. J. Manor D. transfer protein to vitamin Scholar, M. A. N. J. Manor D. of transfer by the transfer Scholar, Manor D. J. Effect of and on transfer by the alpha-tocopherol transfer Scholar, M. J. J. C. Manor D. Vitamin E and the of the α-tocopherol transfer Scholar). vitamin E from astrocytes from that this process is TTP Taken together, these show that trafficking of vitamin E the involves of vitamin E in astrocytes, and secretion from these to neighboring of in cultured cerebellar by and in during of secretion from are the role of in this an of ABC-type to cerebellar astrocytes with as in the our of by and in during of secretion from are the role of in this an of ABC-type to in a cerebellar astrocytes with as in the our of we to which in the transport of vitamin E astrocytes and neurons. are expressed in the brain and trafficking of C. for lipoprotein in the Biophys. Scholar, N. in neurons and Scholar, of the Scholar, J.E. and function of in the nervous Biophys. Scholar, N. C. functions as a efflux Scholar), we role in vitamin E transport in cultured we the secretion of vitamin E in cultured astrocytes in the and of an of ABC-type N. C. functions as a efflux Scholar). We cerebellar astrocytes with and the in as We a to in in with control These indicate that efflux of vitamin E from astrocytes is mediated by a We also to the that vitamin E uptake by neurons. We found that of the low-density lipoprotein receptor–related protein N. in neurons and Scholar, J.E. and function of in the nervous Biophys. in neurons uptake by to Taken together, our that vitamin E of astrocytes in a that TTP and an ABC-type transporter, and that uptake by neurons involves the is to that for the transport of astrocytes and neurons and function in neurons of the central nervous Sci. Scholar, T. D. J. by astrocytes from and human Scholar). is vitamin E trafficking in the nervous system is a process is to by physiological We have that the of the TtpA gene in is by oxidative stress L. C. N. J. Manor D. of the α-tocopherol transfer protein gene is by oxidative stress and Med. Scholar). this also in cerebellar we oxidative stress in and cerebellar with and the of the TtpA We found that of to the a in free as by the which on cellular oxidative stress by Med. Scholar). of the with α-tocopherol the to the antioxidant and the of this cultured cerebellar astrocytes, oxidative stress by a in TtpA which by with vitamin E cultured cerebellar neurons, on the of the TtpA gene to oxidative stress to the of α-tocopherol and that of TTP in to oxidative stress in L. C. N. J. Manor D. of the α-tocopherol transfer protein gene is by oxidative stress and Med. Scholar), T. C. A. stress α-tocopherol transfer protein in human Med. Scholar), an oxidative stress in Scholar), and human brain T. A. M. Kayden H.J. of alpha-tocopherol transfer protein in the of patients with with vitamin E deficiency and oxidative stress neurodegenerative Scholar). The α-tocopherol and neurologic health in by the that mutations in the TTPA gene the familial neurological T. M. T. Y. Y. N. spinocerebellar by a in the gene for the Engl. J. Med. 1995; Scholar, T. T. T. and by a in the gene for the Engl. J. Med. Scholar). The TTP by the TTPA has been to levels of α-tocopherol by the secretion of the vitamin from to M. Kayden M. M. with vitamin E deficiency is by mutations in the alpha-tocopherol transfer 1995; Scholar). has been the neurological of with the that the of of TTP is the of the protein in the We to the role of TTP in vitamin E transport in the We our on the TTP is expressed in this it is the of neurological in it is the brain where the of gene is found in Y. J. L. M.G. profile of stress and neurodegeneration in in alpha-tocopherol transfer Med. and is the most active in vitamin E of vitamin E in and and the uptake of vitamin E by of Biophys. Scholar). We found that, in the cerebellum, TTP is selectively expressed in the astrocytes, where it facilitates efflux of vitamin E to neighboring neurons an ABC-type of vitamin E into neurons is in by the we found that induction of oxidative stress enhances the transcription of the gene in astrocytes, a to oxidative in the brain in the of oxidative TTP is most expressed in patients with neurologic and indicate of the and T. T. J. T. M. M. M. of with by of the alpha-tocopherol transfer protein Scholar, A. M. M. C. M. with vitamin E neuropathological of a 93: Scholar). of the the cerebellum, and is important to that molecular and first in the of vitamin J. M. M. M. and the during alpha-tocopherol deficiency in Med. Scholar). the spinocerebellar as these in by to of L. Manor D. Vitamin E is essential for Scholar, T. T. A. Y. M. in transfer for by chronic oxidative Acad. Sci. A. Scholar). data show that cerebellar of the TtpA gene with the and the of the spinocerebellar and an that of TTP in cerebellar glial A. T. T. M. M. J. of alpha-tocopherol transfer protein in Scholar). the of astrocytes the most and metabolic to neurons, for and uptake of and from the A. N. Rev. Scholar). of vitamin E efflux from astrocytes to neighboring neurons is to transport in the brain P. L. of and in brain the of an in vitro Scholar). secretion of α-tocopherol from astrocytes and uptake of the vitamin into neurons is mediated by the that transport that an ABC-type and the J. and in the Scholar). to transport in the brain to and function of the brain J. J. J. in to brain lipid and and Scholar, R.J. J.D. of function and whole-body Acad. Sci. A. Scholar). data the that vitamin E status is also in these and an important and role in that levels of the in astrocytes in oxidative stress, to observations in J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar). transcription of TtpA in cerebellar neurons by oxidative stress. astrocytes and vitamin TTP in these is to efflux of vitamin E to neurons. of as found in human are to delivery of α-tocopherol to neurons, in oxidative and function that as cerebellar ataxia. from this and to the of vitamin E transport in the that is in transport in the brain is mediated by lipoprotein that are by astrocytes and to to neurons. E deficiency to brain uptake of into Biophys. Scholar, C. E and control over vitamin E in of Scholar, of E vitamin E show in into the Sci. have that vitamin E transport in the brain the with for disease Y. E and for Med. and Alzheimer's disease G.W. M.A. of E and the of Alzheimer's disease in 1993; Scholar), is with vitamin E status P. J.K. of E on and vitamin E Nutr. Scholar). has been that vitamin E of CNS be in Alzheimer's disease N. T.J. E and from protein Scholar). this as uptake of vitamin E into neurons involves the major in the J. J. L. protein regulates brain E and lipoprotein Scholar). levels are in of patients with Alzheimer's disease L. N. of and Alzheimer's disease by the protein Clin. Scholar). the of on in Alzheimer's disease is on M. M. T. Y. T. on its Clin. Scholar), the implications of vitamin with and the of neurodegenerative our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the molecular mechanisms of vitamin E transport in the CNS, which have important implications to the molecular underpinnings of oxidative stress–related neurodegenerative diseases. to the and The by of and of the gene and Y. Morrow J.D. D. D. L. M.G. in in transfer protein and vitamin Acad. Sci. A. Scholar). with and by and in for by for by transcription a for of and to the and for and of in cerebellar the of T.D. of gene data and the Scholar). The and of these J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar, M. P. J. Manor D. of a vitamin E as a for transfer protein 45: Scholar, P. M. Manor D. J. of for in protein and with the alpha-tocopherol transfer Med. Scholar, J. J. Manor D. of mutations in the alpha-tocopherol transfer 45: Scholar, D. J. J. Manor D. transfer protein to vitamin Scholar, M. A. N. J. Manor D. of transfer by the transfer Scholar, C. J. and of form of vitamin Scholar, A. C. M. M. Manor D. J. The of to and transfer by the α-tocopherol transfer protein Scholar). in C. J. and of form of vitamin in a in of and of and in the The to the to and as J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar). in in and of TTP in a against human TTP G.W. L. Manor D. J. M.G. The α-tocopherol transfer protein is essential for from of from the of and Scholar), in and and by with in for a with of and for in and on 50 after to a of and and after the and of the for after to The and by with The cerebellar from by a of for N. P. of for long-term Scholar). and to on a tissue in for and in essential and and cultured for against human and a control in the into human and from and by for The in and to cultured with and after the J. P. J. Manor D. trafficking of vitamin E in The role of transfer into on 50 the in in by with to the for and to in on an to the N. P. of for long-term the of from cultured as J. P. J. Manor D. trafficking of vitamin E in The role of transfer Scholar). in as and from with for and in for the secretion of The secretion and with the from subsequent to the of cellular to the of in the samples and the of astrocytes in for with for with to the free and by after of in the of to the to from in on a of as of to protein in the protein cerebellar on and as for in neurons astrocytes. for neurons and astrocytes after The and in 50 of to on a cerebellar into and cultured for to with The the in in for and with the of for a in on a and to as after with in 50 in the for in a and and with and for data are the The that have of with the of this J. A. and D. M. L. L. M. and L. J. C. and D. M. J. C. and D. M. D. M. D. M. by the of and D. and L. The is the of the and the of the of

Topics & Concepts