Tay-Sachs+Disease+Research+Assignment

Thomas D. Monteverde Biochemistry Prof. Dhar 14 November 2009

__Tay-Sachs Disease (GM2 gangliosidosis or Hexosaminidase A deficiency)__

March 7th, 1881 a twelve month old infant arrived at London Hospital in a severe state of torpor. It was brought to the attention of the hospital staff that the child, three weeks after birth was incapable of holding its head up or moving its limbs and that the child became weaker as time passed. The infant was studied by Warren Tay and no paralysis of any part of the body could be found. Warren Tay determined that the child's cerebral development was deficient and examined the eyes to ascertain whether there was any affection of the optic nerves (Tay, 104). Upon examination Tay noticed that the optic discs were normal, but in the region of the yellow spot in each eye there was a conspicuous, tolerably defined, large white patch, more or less circular in outline, and showing at its center a brownish-red, fairly circular spot, contrasting strongly with the white patch surrounding it (Tay, 104). Warren Tay was unable to reach any conclusion as to the cause of the child's lethargy or the the white patch in the macula of the eye, but suggested that the disease could be congenital. The child was examined again on July 30th, 1881 and it was found that the optic discs had become completely atrophic (Tay, 104). There was little development in what caused the disease in the child and another case study was not reported for several years.

In 1886 Bernard Sachs examined a two year old girl suffering from an odd form of idiocy. The child was born at full term and was well proportioned, but at the age of two months it was observed that the child was lethargic, unresponsive and nystagmic. Families of both the father and mother had histories of mental illness. The child's head, back and neck muscles were too weak to sit upright and hold her head up. All the child's muscles seemed flaccid and as a result she seemed to never attempt any voluntary movement and remained on her back unable to move. The child responded to touch and sound, but was unresponsive to visual stimulation. The child's fovea centralis (central area of the macula region of the retina) was a cherry red color and surrounded by an intense grayish-white opacity and this opacity was most distinct in the vicinity of the fovea centralis and for some distance around it, but faded away gradually into the normal retinal field (Sachs, 542). Sachs determined that the child suffered from an unknown developmental defect. She was studied several months later and it was observed that the optic nerves were completely white with no sign of blood vessels. It was determined that the optic nerves were completely atrophied and the child was totally blind. The child soon deteriorated to the point where she was incapable of ingesting food and died in the month of August.

An autopsy was preformed shortly after the child's death with special attention paid to the skull, brain, and abdominal viscera. On visual inspection the child looked severally emaciated. The skull seemed abnormally thick and the cap was unusually heavy. Abnormalities were also observed in the brain. Their was a large organized clot in the superior longitudinal sinus and the cortex was hard to the touch and had small calcified plates. Numerous longitudinal and vertical sections of both optic nerves were stained and examined, but no morbid changes could be determined which lead to the assertion that ocular degeneration was due to cortical and retinal changes (Sachs, 542). The brain tissue under magnification showed brain cells with absent or distorted nuclei and a peculiar cell mass was present with an odd space surrounding it. The spleen was enlarged and the liver cirrhotic. Its was determined that the disease was hereditary and characterized by arrested cortical development. Bernard Sachs named the disease amaurotic family idiocy due to the familial nature of the disease, but he could not determine the cause of the disease. The disease became known as Tay-Sachs disease for both Warren Tay and Bernard Sachs role in its discovery.

It has been determined that Tay-Sachs disease is an autosomal recessive trait coded by the HEXA gene on chromosome 15. The disease is extremely rare, but occurs in a significant frequency amongst select populations. Carrier frequency among the general population is approximately one in three hundred, but in Ashkenazi Jewish, French Canadian and Cajun populations the frequency of being a carrier is much more significant. One in twenty five Ashkenazi Jews are carriers of the recessive trait (Burton, 2). In order to be affected by the disease both parents must be carriers of the trait and even then there is only a twenty five percent chance of having Tay-Sachs disease. The collective incidence of all lysosomal storage disorders amongst the entire population is approximately one in nine thousand. Due to the recessive nature of the disease only one in twenty seven hundred children are affected by the disease among populations that are significant carriers of the trait, but every child affected dies as a result of the terminal nature of the disease (Burton, 2). Both Tay Warren's and Bernard Sachs' studies of the disease were crucial for the determination of the symptoms that define Tay-Sachs disease, but little was understood as to the physiological causes until the studies preformed by Ernest Klenk in the 1930's and 40's.

In 1935 Ernest Klenk isolated a water soluble glycolipid from the brain of a patient diagnosed with Tay-Sachs disease. The molecule was a new class of carbohydrate rich glycolipid, which he named gangliosides (Svennerholm, 145). Gangliosides are oligosaccharides connected to both a ceramide and one or more sialic acids. A Bial's test was used to determine the concentration of gangliocides in the brain. The Bial's orcinol reaction is the dehydration of either pentose or hexose to form either furfural or 5-hydroxymethylfurfural respectively. Presence of pentose will produce a blue product while hexose will yield a muddy brown-gray precipitate (Schreck, 3). The Bial's orcinol reaction produces a violet color in the presence of glycolipids. It was observed that sialic acid produced the same effect in the presence of Bial's reagent as glycolipids. Using this reagent Ernest Klenk developed a method for quantitative determination of gangliosides (Svennerholm, 147). It was found that a particular ganglioside (GM2) was present in much higher concentrations in brain tissues of patients with Tay-Sachs then in that of normal humans.

In 1968 Robinson and Stirling discovered the presence of beta-N-acetylglucosaminidase isozymes A and B in the human spleen, They were also able to show that the two isozymes were able to cleave p-nitrophenyl-N-acetylgalactoseaminide (Li, 3). This process is crucial in the degradation of gangliosides. The two components were resolved by starch gel electrophorisis (Banerjee, 113). Shortly after the discover of the enzyme, three independent researchers observed that patients with Tay-Sachs disease were deficient in component A of the enzyme (Banerjee, 113). The coincidence between the increased concentration of GM2 gangliosides in the brains of Tay-Sachs patients and the deficiency of Hex A lead to the conclusion that Hex A is the only enzyme involved in the catabolism of of GM2 gangliosides.

Tay-Sachs Disease is a progressive neurodegenerative disorder due to the deficiency either of a hydrolytic lysosomal enzyme or the ancillary activator protein directly responsible for degradation of GM2 gangliosides in the brain (Zeng, 1). Gangliosides are commonly found on the cell surfaces and embedded in the the plasma membrane of cells in the nervous system. Six percent of all phospholipids in the nervous system are gangliosides. As a result brain and nerve cells are affected most severely by this lysosomal storage disorder. The ceramide portion of the ganglioside is embedded in the hydrophobic region of the plasma membrane; while the ogliosaccharide and sialic portions of the gangliosides are exposed to the outside of the cell. Gangliosides function as surface markers that serve as cellular recognition sites and are involved in cell to cell communication. Gangliosides are degraded in the lysosomes in a stepwise manner by interdependent exo-glycosidases (Lemieux, 914). GM2 gangliosides are the primary intermediate in the degradation of GM1 gangliosides. Hydrolytic cleavage of a galactose converts GM1 gangliosides to GM2 gangliosides. The principle role of the lysosomal system is to degrade and recycle substrates derived from the cell surface by endocytosis and from inside the cell by way of autophagy (Walkley, 730). As a result gangliosides build up in the lysosomes which are the waste processing centers of the cell. Lysosomes begin to bulge and eventually burst. The overloading of gangliosides in the cell leads to cell damage. Typical brain tissue of patients suffering from gangliosidosis experience diffuse, foamy, cytoplasmic vacoulation and expansion of the cerebellar purkinje cells, cerebral neurons and astrocytes (Zeng, 61). Neurons balloon and fill with foamy vacuolar and granular lipid material that displaces the cell nucleus. An accumulation of GM2 gangliosides are the cause of untimely cell death and neurodegeneration that is associated with Tay-Sachs disease. Cell fractionation studies of brain tissue from Tay-Sachs examining components in the fraction enriched storage bodies showed one third of the dry weight to be ganglioside and the majority to be GM2 ganglioside (Walkley, 730). There have been studies which suggested in addition to an overabundance of gangliosides, deficiency conditions are also created as a result of lysosomal storage disorders. The accumulation of gangliosides within the cell can create blockage of other cellular functions. The study of the mouse model of lysosomal storage disorder illustrated that blockage of the movement of cholesterol out of the endosomes to the mitochondria prevented the production of cholesterol derived neurosteroid biosynthesis, which has devastating consequences on myelination and action potential propagation (Walkley, 731). The deficiency of myelin which is the the dielectric material surrounding the axon of neurons is a specific symptom of Tay-Sachs disease and one cause of neurodegeneration. Endocytosis, which plays a critical role in integrating a wide variety of signal events affecting many cellular functions is also affected by Tay-Sachs disease. Growth factor receptors are internalized by endocytosis, but ganglioside sequesteration in Tay-Sachs disease results in misplacement of growth factor receptors. The phenomenon of ectopic dendritogenisis is one of the consequences of the misplacement of growth factor receptors associated with gangliosidosis (Walkley, 732).

The degenerative pathway of GM2 gangliosides involves the enzyme beta-hexosaminidase and the GM2 activator protein. The process begins when GM2 activator protein removes GM2 from its membranous environment and presents it to enzyme HexA for hydrolysis within the lysosome (Lemieux, 914). The GM2 activator protein is a 22kDa glycoprotein which acts as a specific cofactor for beta-hexosaminidase A (Hex A) in the hydrolytic conversion of GM2 to GM3 (Wright, 411). In order to present GM2 ganglioside to Hex A the GM2 activator protein must create a water soluble complex with the GM2 ganglioside. This complex is formed through multiple interactions of the activator protein with the non-reducing sugar of the carbohydrate head group, sialic acid and the ceramide tail of the GM2 ganglioside. The GM2 activator protein recognizes the carbohydrate head group and partially inserts into the lipid bilayer of the plasma membrane and extracts the ganglioside for presentation to the Hex A enzyme. A particular structural component of interest in the GM2 activator protein is an eight stranded anti-parallel beta-pleated sheet that leaves an accessible hydrophobic cavity that is suitable for binding the ceramide tail of the GM2 ganglioside (Wright, 417). The hydrophobic ceramide moiety is contained within the lipophilic cavity of the activator protein creating a water soluble complex capable of transport in the cytoplasm. The activator protein opens up the rigid conformation of the ganglioside and makes N-acetyl-galactose and sialic acid accessible to the Hex A enzyme. Without this particular process the degradation of GM2 gangliosides would be impossible and an accumulation of GM2 gangliosides would become apparent in the cell. Mutations of the gene which codes for the GM2 activator protein is one of the many causes of GM2 gangliodosis associated with Tay-Sachs disease. These mutations result in the AB variant of Tay-Sachs disease. There are several possible mutations that can affect the activity of the GM2 activator protein. The synthesis of the mutant activator protein is either blocked at the stage of mRNA or at protein folding in the endoplasmic reticulum (Wright, 418). Three of these mutations involve single amino acid changes that dramatically affect the proteins stability and activity. A mutation of the 138th amino acid from cystine to arginine prevents intracellular transport of the protein out of the endoplasmic reticulum and results in the degradation of the protein before post transitional processing (Wright, 418). This is due to the loss of a disulfide bridge with the cystine at the 112th position that causes dissociation of two secondary structure elements (Wright, 418). The disulfide bridge between both the 138th and 112th cystines is necessary for Hex A recognition. A mutation of the of the 169th amino acid from arginine to proline results in premature degradation of the activator protein. This mutation affects the amino acids hydrogen bonding with the glutamic acid at the 87th position and leads to inter-strand destabilization of the beta-pleated sheet. The bulky proline ring could also be the cause of further conformational changes to the activator protein (Wright, 418). A deletion of the lysine at the 88th position also results in premature degradation in the endoplasmic reticulum. The absence of the lysine at the 88th position has a destabilizing effect on specific regions that are essential for membrane binding. The deletion of this particular amino acid causes a shortening of the apolar loop beta-4 and beta-7 and disturbs the alignment of the beta-pleated sheet. These minor substitutions and deletions of singular amino acids have a devastating effect on the structure and functionality of the activator protein and result in an accumulation of gangliosides within the lysosomes. Mutations of the GM2 activator protein can be a cause of Tay-Sachs disease, but mutations of the Hex A enzyme responsible for the degradation of GM2 gangliosides can also be the cause of the neurodegeneration associated with Tay-Sachs disease.

Hex A is a glycosidase consisting of 4044 residues located in the lysosome. The function of Hex A is to remove the terminal non-reducing N-acetylgalactosamine from the GM2 ganglioside producing GM3 ganglioside. This is preformed through substrate assisted catalysis with retention of configuration to remove the terminal beta-linked N-acetylgalatosamine from the oligosaccharide substrate (Lemieux, 918). There are two distinct active sites present in Hex A, one on the alpha-subunit and one on the beta-subunit. In both active sites there is a glutamate residue which acts as a general acid-base that assists in cleaving the terminal beta-linked N-acetylgalactosamine (Lemieux, 914). An adjacent aspartate residue stabilizes the positively charged oxazolinium intermediate that develops during hydrolysis. In the alpha-active site, alpha-asparagine 423 and alpha-arginine 424 residues promote GM2 ganglioside binding by interacting favorable with the negatively charged sialic acid residue (Lemieux, 914). The hydrolysis of GM2 gangliosides is only catalyzed by the alpha-subunit of Hex A. In the alpha-subunit of Hex A alpha-glutamic acid 323 acts as the general acid base residue for protonation of the glycosidic oxygen atom and alpha aspartate 322 provides the negatively charged carboxylate group that stabilizes the positive charge on the nitrogen of the oxazolinium ion (Lemieux, 918). Several mutations of the Hex A enzyme can hinder it's activity and if GM2 ganglioside hydrolysis goes below the critical threshold of approximately ten percent then GM2 gangliosides begin to accumulate in the neural tissue and lead to neurodegeneration.

The reduction in Hex A activity caused by amino acid substitutions or deletions in the amino acid sequence have various biochemical consequences. The majority of residues involved in Tay-Sachs disease are located throughout domain II of the alpha-subunit, distributed amongst the beta-strands and helices comprising the TIM barrel with only a select few mutation occurring among residues of the active site (Lemieux, 920). Amino acid substitutions and deletions can result in several possible outcomes. The loss of a salt bridge, disruption of a beta-sheet, over packing of residues, loss of hydrogen bonding, decrease in hydrophobic interactions and buried polar residues are all consequences of disruptions of the amino acid sequence. Chemical chaperones such as NAG-thialozine can prevent misfolding of the Hex A enzyme mutations associated with adult onset Tay-Sachs disease and can increase the residual activity of Hex A above the critical threshold for neurodegeneration, but numerous other mutations cannot be stabilized by NAG-thialozine (Lemieux, 913).

There has yet to be a cure for Tay-Sachs disease, but there have been several theories for possible cures. The study of animal models, in particular mice have lead to developments of possible cures for Tay-Sachs disease. In the 1990's it was observed that the phenotype of the mouse model of type B Tay-Sachs disease was much milder then that of humans. This observation was found to be due to a difference in GM2 ganglioside catabolism and in particular the GM2 activator protein. In mice the GM2 activator protein could effectively stimulate the hydrolysis of asialo-GM2 also known as GA2 by Hex A and to a lesser extent by Hex B. GA2 is created when the enzyme sialidase removes the sialic acid from GM1 ganglioside. Further catabolism of GA2 is not possible in the human body and as a result GA2 is also commonly found in abnormal concentrations in patient suffering from Tay-Sachs disease. The ability of mice to preform an alternate pathway in the degradation of GM2 gangliosides is due to a small five amino acid sequence on the GM2 activator protein. Replacement of the corresponding site in the human sequence with the amino acid sequence of the mouse converts the ineffective human GM2 activator protein into a chimeric protein capable of preforming the alternate catabolic pathway (Li, 13). This process may be able to be applied to patients with Tay-Sachs disease enabling the further degradation of gangliosides through the GA2 route. More applicable methods have been applied in recent years to mitigate the affects of Tay-Sachs disease, especially in populations that are at a higher incidence rate.

Heterozygous carrier screening is common among populations that have a high frequency of Tay-Sachs disease and has become a common method for Tay-Sachs disease prevention. The study of the genetic mutations of Tay-Sachs disease has made it possible to distinguish through genetic screening who is in fact a heterozygous carrier of a trait associated with a mutation of either the GM2 activator protein or the Hex A enzyme. Since the availability of carrier testing, the incidence of Tay-Sachs disease in Ashkenazi Jews in the United Kingdom has halved (Burton, 4). This process is helpful in the prevention of Tay-Sachs disease, but there is no cure or alleviation of symptoms for homozygous carriers of acute Tay-Sachs disease.

Tay-Sachs disease is a relatively insignificant disease affecting only a minuscule portion of the world population, but the discovery and study of the disease has illuminated the function of several biochemical process of both human and animal physiology. By observing the deficiencies of Tay-Sachs patients it is possible to elucidate the function and processes of several biochemical structures. Tay-Sachs disease played a crucial role in the determination of Hex A function in ganglioside catabolism and has been the muse of recent developments in chimera genetics. NAG-thiazoline has shown to increase the activity of some forms of mutant Hex A enzyme in the chronic adult variant of Tay-Sachs disease, but has been unsuccessful in other acute forms. The recent developments in carrier screening has decreased the occurrence of the disease, but it still affects select populations. The obvious form of possible prevention of Tay-Sachs and most other lysosomal storage disorders for the populations at most risk would be to procreate with a person of differing genetic background. __References__ Bannerjee, D. K., Basu, D. Purification of Normal Human Urinary N-acetyl-beta-hexosaminidase A by Affinity Chromotography. // Biochemistry Journal //, 145(1), 113-118. Burton, H., Levene, S., Alberg, C., et al. Tay-Sachs Carrier Screening in the Ashkenazi Jewish Population: A Needs Assessment and Review of Current Services. // Phg Foundation, // (2009), 1-11. Lemieux M. J., Mark B. L., Cherney M. M., et al. Crystallographic Structure of Human Beta- Hexosaminidase A: Interpretation of Tay-Sachs Mutations and Loss of GM2 Ganglioside Hydrolysis. // Journal of Molecular Biology //, 359(2006), 913-929 Li, Y-T., Li, S-C. Catabolism of GM2 in Man and Mouse. // International Congress Series //, 1223(2001), 3-15. Sachs B. On Arrested Cerebral Development with Special Reference to Cortical Pathology. // Journal of Nervous Mental Disease //, 14(1887), 541-554. Svennerholm, L. The Gangliosides. // Journal of Lipid Research //, (2009), 145-155. Tay W. Symmetrical Changes in the Region of the Yellow Spot in each Eye of an Infant. // Transactions of the Opthalmological Society //, 1(1881), 55-57. Walkley S. U., Vanier M. T. Secondary Lipid Accumulation in Lysosomal Disease. // Biochimica et Biophysica Acta //, 1793(2009), 726-736. Wright C. S., Li S. C., Rastinejad F. Crystal Structure of Human GM2-Activator Protein with a Novel Beta-Cup Topology. Journal of Molecular Biology, 304(2009), 411-422. Zeng, B. J., Torres, P. A., Viner, T. C., et al. Spontaneous Appearance of Tay-Sachs Disease in an Animal Model. // Molecular Genetics and Metabolism //, 95(2008), 59-65.