Therapeutic+effects+of+Glatiramer+Acetate,+Copaxone,+in+Alzheimer's+Disease+Experiments

=Therapeutic effects of Glatiramer Acetate, Copaxone, in Alzheimer's Disease Experiments=


 * Brian Leung **
 * Department of Chemistry **
 * Drexel University **
 * CHEM 367 **


 * Brief History of Alzheimer’s Disease:**

Neurodegeneration and neurogenesis has been a popular research interest in the past few decades. Specifically in the realm of neurodegeneration, Alzheimer’s disease (AD) was first characterized in 1906 by a physician named Alois Alzheimer. Alois Alzheimer first documented this disease when a 51-year-old woman presented to him her cognitive impaired symptoms and died shortly with this strange neurological disease. He described the post mortem morphology of the brain to be shrunken with wide sulci and the morphology of the neuron to have fibrillary tangles in the soma and axons (Berchtold & Cotman, 1998; Vesterberg, 1993). Behaviorally, Emil Kraepelin first documented the characteristics as a special subset of senile dementia. In its early stages, this disease was described to be indistinguishable from senile dementia, until a few decades ago; this disease’s proper name is Senile Dementia of Alzheimer’s Type (SDAT)(Vesterberg, 1993).

Today, AD affects more than 5.3 million people and is the 5th leading cause of death. Commonly, this disease is one of the many comorbid diseases found in one patient, typically 65 years and older. To stress on the severity of this disease, there is no known cure or effective treatment for AD. There are treatments to delay the inevitable fate of AD; however, more work is needed in this field. Furthermore, within the next two years, the first set of baby boomers’ will reach their 65th birthdays, the age where the chances of acquiring AD become significantly higher (Alzheimer's Association, 2009; Nelson, et al., 2009).


 * Pathogenesis of AD:**

On a molecular level, two hallmarks that distinguish AD from other neurodegenerative disease are the presence of amyloid beta (Aβ) and tau neurofibrilary tangles (Reys, et al., 2008; Funke, Birkmann, & Willbold, 2009). Aβ production causes localized axonal deterioration and death of both neurons and oligodendrocytes via caspase mediated apoptosis(Jantaratnotai, Ryu, Kim, & McLarnon, 2003). Although there is a rare form of Alzhimer’s Disease that may only have neurofibrilary tangles but no amyloid plaques(Peters, et al., 2009). Specifically Aβ is produced after proteolytic cleavage of an endogenous, transmembrane protein, amyloid precursor protein, APP. APP is located on chromosome 21, transcribed, translated, modified, and transported to the cell surface (Saunders, Kim, & Tanzi, 1999; Saunders & Tanzi, 2003). At the surface, there are metallo-proteases called α-secretase, β-secretase, and γ-secretase. Enzyme cleavage determines weather Aβ is produced: the amyloidgenic and the non-amyloidgenic. In the non-amyloidgenic pathway, α-secreatase and γ-secretase cleave APP at the extracellular membrane portion just before the transmembrane domain and within the transmembrane domain of APP respectively. This causes the formation of soluble APP (sAPP) that is released into the extracellular matrix: ectodomain shedding. On the other hand, in the amyloidgenic pathway, β-secretase cleaves further in the ectodomain, closer to the N-terminus, and γ-secretase cleaves as well. This cleavage forms an additional product, Aβ 40-42. Aβ causes inflammatory responses and activates other pathways that cause neurodegeneration.


 * Glatiramer Acetate:**

Glatiramer Acetate (GA) is a random polypeptide of 4kda to 16kda; GA was first discovered in 1960 and developed by TEVA pharmaceuticals, an international pharmaceutical company based in Petah Tikva, Israel with branch in North Wales, PA and Kansas City, MO (Schrempf & Ziemssen, 2007). This Pharmaceutical specializes in drugs that treat neurological diseases. The formation of Glatiramer Acetate is divided into four stages: addition of protection sites on side chains and with simultaneous synthesis; elimination of protection species from side chain sites; dilution and ultra purification; lyophilization (TEVA, 2009; Konfino, Sela, Teitelbaum, & Arnon, 1989; 2000; 2002; 2002; 2003; 2005; 2007)

The first step includes the copolymerization of the four anahydrous amnio acids at 25°C with 1,4-dioxane as a solvent for about 24 hours. Furthermore an indicator, diethylamine, is used to yield four protected amino acids. Water is then added to precipitate, chop and disperse them for 1.25 hours with subsequent filtering and washing. The filtrate is then dehydrated at 60°C at 20mmHg for 12 hours before milling. At this stage, the amino acid sequence is fixed and protected from further side chain reactions. The polypeptide is currently has a molecular weight of around 16,000daltons.

The second stage, removing the protecting groups, consist of treating the polypeptides in a solution of 33% HBr and glacial acetic acid at 20°C. The total time interval goes is 18 hours total with a temperature range of 17°C-21°C; step 1 is 15 hours at 18°C and step 2 is 3 hours more after stage 1 at ~20°C. The water is added to was the product with subsequent decanting and filtration and drying at 40°C at 40mmHg. The product currently has a trifluoroacetyl groups and has a lower molecular weight than the product after stage 1, due to the removal of the protecting groups.

The next stage, stage 3, treats the trifluoroacetyl amino acid product with aqueous piperidine at 20°C in a basic solution. The amino acid – now has the trifluoroacetyl products removed – is filtered at 15°C. Ultrafiltration and dilution cycles to remove the low weight polypeptides and the piperidine by acidifying the product with glacial acetic acid to purify the sample to an acceptable level. At this stage is the aqueous version of GA in solution.

The last stage is the lyophilization stage. However, before lyophilization, the product is once again re-filtered with pores at .2um. The lyophilization process occurs at -50°C at a very low pressure at less than .03mbar. The resulting material is described to be from white to slightly yellowish and is the salt form of GA by having a molecular weight range with an HPLC peak at 16,000 – 2,500 daltons (GC-HPLC calibrated with standard polypeptide ladder) (Konfino, Sela, Teitelbaum, & Arnon, 1989; 2000; 2002; 2002; 2003; 2005; 2007).

Other instrumental data for the structure of GA are noted below:

7.09 Tyr aromatic protons; 7.79 Tyr aromatic protons; 4.1 Amino acid alpha protons: 2.96 Lys, 2.27 Glu, 1.87 Acetate || 16.4 β-Ala; 22.7 γ-Lys; 23.8 acetate CH3; 26.8 δ-Lys ca. 28 β-Glu; 30.6 β-Lys; 34.1 γ-Glu ca.; 36 β-Tyr; 39.6 ε-Lys ca.; 51 α-Ala ca.; 55 α-(Lys, Glu, Tyr); 115.9 3'-Tyr; 128.2 1'-Tyr; 131.0 2'-Tyr; 155.1 4'-Tyr ca.; 175 amide carbonyls; 181.7 δ-Glu || Characteristic of alpha-helix region || 1550.6 N--H in-plane bending modified by C--N stretch; 1406.0CO2'' symmetric vibration 1248.1 C--N stretching mode modified by N--H in-plane bending (amide III) || 0.427 alanine, 0.337 lysine, 0.141 glutamate, and 0.093 tyrosine || Minima 208 nm and 220 nm (alpha-helix) ||
 * = Instrument ||= Important Features of GA ||
 * = Proton NMR ||= In Deuterium oxide soln:
 * = Carbon-13 NMR ||= 300.1 for protons & 75.5MHz
 * = UV spectrum ||= Max Molar Absorptivity value determined at 220nm;
 * = IR ||= 1655.0 C=O stretching (amide I) (alpha-helix);
 * = Edman Degredation ||= Due to random polymerization (molar ratios):
 * = Circular Dichroism (CD) ||= Buffer PBS ph 6.8 with KCl:


 * MS and EAE:**

In many GA studies, this compound has been seen to treat the symptoms of experimental autoimmune encephalomyelitis (EAE) in mice, an experimental version of the disease multiple sclerosis (MS). MS is an idiopathic, inflammatory, demylenating disease that eventually causes neurodegeneration in the white matter of the CNS by creating large lesions that alter normal neuron communication (SITE). Pathological hallmarks of MS include the demylenating plaques and axonal damage (Schrempf & Ziemssen, 2007). MS pathological stages include the relapsing remitting MS (RRMS), Primary Progressive MS (PPMS), and secondary progressive MS (SPMS) (Schrempf & Ziemssen, 2007). Most clinical trials for GA treatment have been conducted during the RRMS stage of MS because the experiments that are performed animals with MS-like pathology showed a higher correlation with the RRMS stage than with the other stages because MS-like pathological animals had MS that was prolonged. Johnson and his team have been doing clinical studies that show how GA decreases MS relapse symptoms by 29% (Johnson 1995).

The most common and well known MS-like pathology that is used in the majority of MS animal studies is EAE. EAE is induced by injecting myelin oligodendrite protein (MOG), a peptide similar to myelin basic protein MBP, which resembles GA. This injection induces the inflammatory response causing neurodegeneration in the CNS and PNS as well as axonal generation in the CNS and PNS. Physiological changes in MS are described as lack of motor control and functionality that can occur acute temporary. (Aharoni, Arnon, & Eilam, 2005)

In MS patients, the mechanism behind neuroprotection and neurogenesis during GA treatment and post GA treatment has been explained for GA; however, the entire mechanism still is not laid out in detail. Current opinions concur that GA has been the treatment that decreases inflammation, suppresses the immune response, and increases neurogenesis. Pre GA injection from imaging techniques show that T helper cells (TH) cross the blood brain barrier (BBB) into to CNS and become activated when myelin binds on to the antigen binding cells APC, such as microglia. This induction promotes the TH to secrete proinflammatory cytokines and chemotaxins to the specific sites in the CNS. The inflammatory pathway activates and promotes macrophages and other endocytic cells to engulf any foreign bodies. Furthermore, it also activates B cells to secrete anti myelin antibodies (AB) that destruct the myelin sheath. This entire inflammatory response damages neurons causing the damage mostly be irreversible or reversible. Although most experiments GA treatments performed in EAE, the similarities of both demyelinating diseases generally do agree on many pathways.GA targets the TH in both diseases and immunomodulates other inflammatory cells: CD8+ cells, B cells, and APCs (Murphy, Travers & Walport, 2008).


 * Neurogenesis:**

Past beliefs about adult neurogenesis was once phrased as impossible because neurogenesis was once perceived to only occur during neural devlopment; however, this phenomenon was discovered in 1992 when Reynolds and his team discovered in a mammalian brain that houses neural multipotent cells or Neural stem cells (NSCs): subvetricular zone (SVZ) and subgrannular Cells (SGC)(Reynolds & Weiss, 1992). These proliferating cells were visualized via immunofluorescence and //in situ// hybridization of fixed neuronal slices. These fluorescent markers such as bromodeoxyuridine (BrdU), a nucleoside used to intercalate between the hydrophobic nucleotide layers of DNA during S phase.These multipotent cells were visualized and seen as a strip migrating from the SVZ and SGC to other cortical areas through the rostral migratory stream (RMS) and towards the olfactory bulb (OB) (Reynolds & Weiss, 1992). These progenitors are capable of differentiating into neurons and other gilal cells whenever needed and are regulated upon the release of neurotransmitters gamma butyric acid (GABA) and substance P (Miranda-Contreras, Benitez-Diaz, Pena-Contreras, Mendoza-Briceño, & Palacios-Prü, 2002; Reynolds & Weiss, 1992). Once activated, these neural progenitor cells migrate through the RMS towards the OB (Reynolds & Weiss, 1992; Bear, Connors, & Paradiso, 2007). However, there are some instances where neurons may migrate away from the RMS and into lesions formed by neurodegenerative diseases to wherever they are needed or to locations in the brain that are stimulated more (Aharoni, Arnon, & Eilam, 2005; Bear, Connors, & Paradiso, 2007).


 * Neuroprotection and Neurogenesis with GA Treatment Mechanisms:**

In MS patients, the mechanism behind neuroprotection and neurogenesis during GA treatment and post GA treatment has been explained for GA; however, the entire mechanism still is not laid out in detail. Current data suggests that GA has been the treatment that decreases inflammation, suppresses the immune response, and increases neurogenesis. Pre GA injection from imaging techniques show that T-helper cells (TH) cross the blood brain barrier (BBB) into to CNS and become activated when myelin binds on to the antigen binding cells APC, such as microglia. This induction promotes the TH to secrete proinflammatory cytokines and chemotaxins to the specific sites in the CNS. The inflammatory pathway activates and promotes macrophages and other endocytic cells to engulf any foreign bodies. Furthermore, it also activates B cells to secrete anti myelin antibodies (AB) that destruct the myelin sheath. This entire inflammatory response damages neurons causing the damage mostly be irreversible or reversible. Although most experiments GA treatments performed in EAE, the similarities of both demyelinating diseases generally do agree on many pathways.GA targets the TH in both diseases and immunomodulates other inflammatory cells: CD8+ cells, B cells, and APCs (Schrempf & Ziemssen, 2007).

Current mechanisms for how GA interacts with the immune system also interact with the nervous system. The mechanism of GA beings by binding to the major histocompatability complex class II (MHC) of antigen presenting cells (APC). With APC, GA interacts with the receptors more efficiently than MBP and also bind to the antigen-binding site for MBP AB in both the PNS and CNS. This competition decreases the MBP binding the AB and decrease in MBP loss. This difference in binding causes a decrease in tumor necrosis factor – α (TNF-α), a decrease in capthesin B, and an increase in interleukin (IL-10). Upon APC binding to GA, this activates bone marrow cells to differentiate into dendritic cells due to IL-10. Furthermore, there is a conversion of these dendritic TH1 cells to TH2 cells. This shift is caused by a decrease in IL-4, a decrease in TNF-α, and a decrease in Interferon-γ (INF-γ); all of those are responses and promotes anti-inflammation. Whereas in MS and EAE studies, the initial levels of IL-4, TNF-α, and INF-γ are increased. Note that most of these pathways are identified //in vivo//. Many scientists have agreed upon the fact that GA is unlikely to cross the BBB at high concentrations, instead the GA-TH2 cells are reactivated in the CNS and cross react with the body’s own myelin causing this anti-inflammatory response. This anti-inflammatory response allows the TH1, TH2, TH0 to all secrete brain derived neurotrophic factor BDNF and other neurotrophic factors. Furthermore, B cells are also activated by TH2 cells and secrete anti GA Abs. These ABs negatively affects the GA treatment in vitro but in vivo, more experiments need to be performed to confirm an effect in vivo. Although these many are beneficial responses for GA treatment in MS and EAE, this immunosuppressant just suppresses these symptoms, not eradicating them (Schrempf & Ziemssen, 2007).


 * AD Inflammatory Response Aβ:**

AD Pathology with it is a fact that inflammatory responses occur in AD and there are copious amounts of data that support that microglia and other immune cells clears amyloid beta. The question now is, why does AD progress despite amyloid beta clearance?There are many theories that are accepted and some that are scrutinized. Some theories describe the neurons as being overwhelmed with the increase the concentration of Aβ due to an increase in production and a decrease in clearance. Some other theories describe aging microglia being not as effective. However, some theories are shown in literature to be true, while others still need more data. One study suggests that aging microglia become dysfunctional and lose their ability to degrade Aβ and recognize Aβ. Recognition of Aβ decreases because these microglia can not express Aβ binding receptors. What is peculiar is that proinflammatory chemokines are upregulated: IL-1β and TNFα. Ironically, TNFα is responsible for the downregulation of Aβ recognition receptors, SRA and CD36, on microglia.(Hickman, Allision, & El Khoury, 2008).

Aβ recognition binding to the MHCII on any macrophage or endocytic cell will express increasing levels of proinflammatory cytokines IL-1β, IL-6, TNFα, IL8 and other proinflammatory mediators. These cytokines promote migration of monocytes, macrophages, and microglia to the site of localized Aβ (Rogers & Lue, 2001; Hickman, Allision, & El Khoury, 2008). However, there is some overwhelming data suggesting that these proinflammatory there is also some data that suggests that Aβ binds to a surface protein called receptor for advanced glycation endproducts (RAGE). This receptor is expressed in neurons, microglia and endothelial cells. Once bound, the transcription factor NF-kB is upregulated as well as oxidative stress. Furthermore, another compound, macrophage colony-stimulated factor (M-CSF), is produced unregulated at a certain locus in the CNS. (Lue, et al., 2001). In addition, aging TH increase the concentration of amyloid beta in the CNS via reducing TH signaling intensity, reduced proliferation of TH receptor stimulation, decrease conversion from TH1 to TH2 (causing more inflammation than immunomodulation), and decreased cognate helper function; all the above reduced regulations correlate with Alzheimer’s-like pathology seen in vivo (Maue, Yager, Swain, Woodland, Blackman, & Haynes, 2009; Haynes & Maue, 2009).


 * GA treatment with AD:**

There is overwhelming scientific evidence that describes GA as having neuroprotective effects and indirectly promotes neurogenesis in the CNS. The literature suggests that GA follows almost the same inflammatory response by activating TH and microglia to induce neuroprotective effects and neurogenesis in the CNS. Unlike MS, where the T Cells react with MBP, AD pathology reacts with Aβ. This interaction with Aβ is crucial because it shifts the neurodegeneration to neuroprotection causing Aβ degradation(Butovsky, et al., 2006). Data for neuroprotection suggests that microglia destroys Aβ neurons caused by an upregulation of CD14 from neurons(Bate, Veerhuis, Eikelenboom, & Williams, 2004; Frenkel, Maron, Burt & Weiner, 2005). If Aβ is downregulated, the concentration ofCD14 would not cause neuronal death via microglia. During GA treatment, GA activates the growth of hippocampus neurons by inducing insulin-like-growth factor-1. The mechanism for expressing insulin-like growth factor 1 is by causing a phenotype switch in the microglia (Schaeffer, Novaes, da Silva, Skaf, & Mendes-Neto, 2009; Butovsky, et al., 2006). This neurogenesis is most likely caused by the same pathway as in MS/EAE studies; furthermore, and as mentioned by Hickman et al., the microglia are not efficient enough to phagocytize the amyloid beta present in the CNS (Butovsky, et al., 2006)(Hickman, Allision, & El Khoury, 2008). Suggested and confirmed regulators have been proposed to describe the regulation of microglial activation specifically in AD. IL-4 concentrations have been shown to modulate Aβ-microglia, not INF-γ.

During GA activation, TH1 cells morph to become TH2 cells. The recruitment of more TH2 cells or TH cells are located in the bone marrow and activated upon chemotaxins secreted by TH cells in the CNS and chemotaxis to specific location. Selective ablation methods to remove bone marrow were to confirm the initial location for where migrating dendritic cells were located at before recruitment post GA treatment. These bone marrow cells (BM-cells) were identified via immunohistochemistry with transgenic mice. Thus the increase concentration of TH2 cells are reconfirmed in AD mice to show that the pathways as to dendritic recruitment are similar, but more research needs to be performed (Butovsky, Kunis, Koronyo-Hamaoui, & Schwartz, 2007).


 * Conclusion:**

Alzheimer’s Disease pathology is caused by the improper protein formation of due to improper tau protein nitration and improper APP proteolytic cleavage. This disease causes localized neurodegeneration via inflammation induced by amyloid beta. Current data has shown that a polypeptide, glatiramer acetate, is a crucial drug used for neuroprotection and neurogenesis via immunomodulation in MS patients and EAE animal models. Because MS and AD are both neurodegenerative diseases and the antigen causes a similar inflammatory pathway, GA treatment in MS has been shifted to see its effectiveness as an AD treatment. Since GA treatment in AD experiments have shown that inflammation essential for neuroprotection and neurogenesis, the current scientific community has agreed that the treatment for AD should not be treated as an immunosuppressant but rather immune activation.


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