The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.

[1]  S. Narumiya,et al.  Expanded polyglutamine in the Machado–Joseph disease protein induces cell death in vitro and in vivo , 1996, Nature Genetics.

[2]  A Dürr,et al.  Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions. , 1998, Human molecular genetics.

[3]  A. Brunet,et al.  Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1). , 1994, The EMBO journal.

[4]  Masaaki Nakamura,et al.  Exonic trinucleotide repeats and expression of androgen receptor gene in spinal cord from X-linked spinal and bulbar muscular atrophy , 1994, Journal of the Neurological Sciences.

[5]  O. Onodera,et al.  Unstable expansion of CAG repeat in hereditary dentatorubral–pallidoluysian atrophy (DRPLA) , 1994, Nature Genetics.

[6]  C. Sherr,et al.  Type C viruses of baboons: isolation from normal cell cultures. , 1974, Cell.

[7]  A. Sano,et al.  Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p , 1994, Nature Genetics.

[8]  Georg Auburger,et al.  Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2 , 1996, Nature Genetics.

[9]  Y. Agid,et al.  Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion , 1997, Nature Genetics.

[10]  A. Roses,et al.  Polyglutamine Domains Are Substrates of Tissue Transglutaminase: Does Transglutaminase Play a Role in Expanded CAG/Poly‐Q Neurodegenerative Diseases? , 1997, Journal of neurochemistry.

[11]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[12]  K. Fischbeck,et al.  Intranuclear Inclusions of Expanded Polyglutamine Protein in Spinocerebellar Ataxia Type 3 , 1997, Neuron.

[13]  H. Green,et al.  Transglutaminase action imitates Huntington's disease: selective polymerization of Huntingtin containing expanded polyglutamine. , 1998, Molecular cell.

[14]  Yves Agid,et al.  Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats , 1996, Nature Genetics.

[15]  C A Ross,et al.  Intranuclear Neuronal Inclusions: A Common Pathogenic Mechanism for Glutamine-Repeat Neurodegenerative Diseases? , 1997, Neuron.

[16]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[17]  D. Housman.  Gain of glutamines, gain of function? , 1995, Nature Genetics.

[18]  S. Tsuji,et al.  Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch , 1998, Nature Genetics.

[19]  R. Lauro,et al.  Transglutaminase activity is related to CAG repeat length in patients with Huntington’s disease , 1996, Human Genetics.

[20]  Harry T. Orr,et al.  Identification and characterization of the gene causing type 1 spinocerebellar ataxia , 1994, Nature Genetics.

[21]  L. Lorand,et al.  A filter paper assay for transamidating enzymes using radioactive amine substrates. , 1972, Analytical biochemistry.

[22]  Andrew R. Cherenson,et al.  The structure of an antigenic determinant in a protein , 1984, Cell.

[23]  Shigenobu Nakamura,et al.  CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1 , 1994, Nature Genetics.

[24]  S. Ōmura,et al.  Lactacystin, an inhibitor of the proteasome, blocks the degradation of a mutant precursor of glycosylphosphatidylinositol-linked protein in a pre-Golgi compartment. , 1996, Biochemical and biophysical research communications.