This review article to determine the activation of telomerase enzyme on neuron cells and how it is used as therapeutic target to treat aging of neurons on disease condition, animal models experimental and case studies such as activate telomerase enzyme to preserve telomere gene length, preventing telomere shortening on age related disease especially in aging of neurons which impacting on age related neurodegenerative disorders.
Mice brain astrocytes models were used with inducing telomerase enzyme (TERT) expression with hypoxia and ischemia condition, the results shows that how it has impacted on neuronal apoptosis, neuronal survival, regulating self-renewal mechanism to recover from the brain injury, these studies helps to improve the therapeutic targets t treat age related diseases and also helps for further future experiments to providing broad health improvement. Experiment results were shows that TERT expression regulates astrocytes proliferation which helps on recover of hypoxia ischemia condition on mice brain, furthermore TERT expression regulates growth factors, cell regulatory proteins which are responsible on cell division and proliferation of cells.
Aging is a biological process and natural selection which affect most of cells, organisms and species which is the major complication of some of diseases such as cancer, cardiovascular diseases and neurodegenerative diseases. Telomeres are the biological clock, shortening of telomere parallel with aging of cells. Overall experimental evidences suggests that telomere shortening, un capping and cellular senescence results as aging, researchers monitoring large population telomere length and telomerase activity measurements at multiple times to proven relationship of initiation and progression of age associated diseases, generally the researches and progressing treatments focused to eliminate the cause of aging to prevent the prevalent diseases. In the review discuss about the role about the telomere activity on aging associated neurodegeneration diseases, does activation of telomerase enzyme regrow or renew the neuronal cells, aspect to prevent aging associated neurodegenerative diseases of central nervous system such as stroke, epilepsy, Parkinson’s disease, Alzheimer’s disease, and other dementias (Ferron, 2009).
Central nervous system consist of brain and spinal cord which are made up of two type of cells such as neurons and glia, neurons transfer electric nerve impulses and chemical signals of brain and central nervous system, glial cells also called as astrocytes and oligodendrocytes. Neuron made up of 3 basic structures such as cell body, axons, and dendrite. Cell body has nucleus which responsible for nerve cell activities, axon has elongated finger like projections which can transmit message from receiver cell to central nervous system or transfer nerve impulse from central nervous system to transmitter cells, dendrites appears like tree branches which receiving nerve impulse for the cell, neurons are interconnected through synapses and neurotransmitter which are chemical signal transmitter between the two neuronal axons or dendrites.
There are 3 types of neurons such as sensory neurons, motor neurons, intermediate neurons. Sensory neurons carries stimuli nerve impulse from receiver or sensory organs to the central nervous system, intermediate neurons present as intermediate between sensory neurons and motor neurons , which receives nerve impulse from sensory neurons and transfer to motor neurons. Motor neurons transfer nerve impulse away from central nervous system to effector cells.
Neurons are generated by neural stem cells which has ability of self-re-new and producing more stem cells or progenitor cells, stem cell could differentiate into different type of cells either into neurons or astrocytes or glial cells. Basically neuron cells are long life cells but also die due to many reasons such as during migration and earlier differentiation, progressive neuronal loss, reduced levels of neurotransmitters, inflammation, loss of integrity of central nervous system, infraction.
Researches had current research to manipulate the stem cells to recover from aged neuron cells or diseased neuron cells, generally neurological disorder research conducted by National Institute of Neurological Disorders and Stroke and Institute Brains Resources and Information Network (BRAIN , they undertaken research to produce growth factors and other signaling mechanisms to activate precursor cell to re grow new neurons.
Role of telomere and telomerase enzyme in aging of neuron cells
Telomere are located at the end of chromosomes made up of tandem repeats of DNA sequences complexed by specialized six protein complexes which is called as shelterins such as TRF1, TRF2, POT1, TIN2, TPP1, and RAP1. TRF1 and TRF2 bound to double stranded telomeric repeats (Donner, (1991)), POT1 bind along with single stranded telomeric repeats on DNA cap, TIN2 bounds with TRF1 and TRF2 and also re organized as TPP1-POT1 hetrodimer, RAP1 is complexed with TRF2 (Donner, (1991)), all together formed as telomere cap, telomere stabilized as structure as protective T loop structure, telomere DNA sequences made up of repeated sequences of 5’-TTAGGG-3’ as 150 to 2000 times. Telomere plays significant role in cell division which regulate controlled and possible cell division, regulate transcription, and protect the chromosomes. Telomere is biological clock as mentioned which is determining the possible cell division which is known hay flick’s limit where the cell divides and terminated due to shortening of telomere in length, telomere is shortened as length of 50-200 base pairs during each cell divisions.
Telomerase ribonucleoprotein complex which is DNA reverse transcriptase polymerase also referred as telomerase reverse transcriptase(TERT) which switched on during once in lifetime to produce telomeric DNA sequences on top of chromosomes by using RNA template, telomerase enzyme activation or expression prevented after embryonic development but telomerase expressed on cancer which results as expending the length of telomere which results as abnormal cell division , telomere got shortening through the lifespan of organism due to the prevention of the activation of telomerase enzyme which results as physiological aging and also related aging of neuron cells to cause neurodegenerative diseases.
Genetic Epidemiology Research on Adult Health and Aging (GERA) conduct epidemiological studies around 100,000 individuals about telomere shortening how effect on the aging to get knowledge about therapeutic target of telomere shortening and preventable methods experiment on other species like mice, this organization also discover longitude changes of telomere also impact on aging of neuron cells and established that telomere shortening is biomarkers or hallmark of aging of cells.
Telomerase enzyme focused to use as potential therapeutic method to increasing longevity of neuron cell to prevent or treat age related neurological diseases.
Aging related neurological diseases association with telomere length
Telomere length shortening directly involves in reduce the longevity of neuron cells and aging of neuron and glial cells to cause neurodegenerative diseases but also indirectly involves on progression on neurodegenerative diseases due to altered the neuronal stem cells by impaired with the self re newel mechanism of central nervous system cells which leads to loss of self re newel mechanism of neurons and glial cells to prevent from neurological diseases (Bär, 2016).
There is recent research conducted to identified correlation of telomere length with longer life span and not affected by any of aging related neurological diseases, they examined telomere length of human subjects from postmortem brain tissue samples which reported as that increased telomere length in older individuals has longer life span and preventable from neurological diseases (Bär, 2016), individuals has short life span and potential to affect by neurological diseases who has shorten telomere length. Telomere length altered due to oxidative stress and inflammation occurs in Alzheimer’s disease, Parkinson disease and Huntington disease.
Alzheimer’s disease is progressive dysfunction and degeneration of neurons in brain regions which is aging related neurodegenerative disorders, it is a main cause of dementia which is characterized as loss of cognitive functions such as thinking, memory ability, behavioral abilities, Alzheimer’s characterized by aggregation of fiber plaques and tangles, plaques formed due to aggregation of amyloid b peptide of neuron cells, tangles formed due to accumulation of hyper phosphorylated Tau proteins formed inside the neurons.
arkinson’s diseases characterized as the dysfunction and degeneration of neurons in the brainstem, substantia nigra and cerebral cortex, loss dopaminergic neurons and imbalance of dopamine and acetylcholine neurotransmitters results as abnormal voluntary muscle contraction.
There are is a test conducted to examined hypoxia ischemia conditioned mice brain which identified as astrocytes (glial neuronal cells) proliferation increases due to the expression of telomerase reverse transcriptase (TERT), researchers examine the rat with hypoxia and ischemia where the astrocytes function or neuronal survive with expression of telomerase, they activate and inhibit the TERT in vivo by gene transduction. Proliferation of astrocytes was examined by Ki67 staining, neuronal injury due to brain infract detected by terminal deoxynucleotidyltransferase- mediated Dutp nick end labelling staining.
Sprague- Dawley rats are used in this research treating hypoxia and ischemia condition on their brain by express the TERT which detected by reverse transcriptase polymerase chain reaction through western blot, and immunohistochemistry.
Cell culture and gene transduction
TERT expression was determined how impact on neuronal survival on mice hypoxia and ischemia injury therefore astrocytes and neurons prepared on cell culturing method , embryonic stage cortical tissues astrocytes and neurons designed with upregulating TERT expression to repair the injured brain cells during HI ( hypoxia ischemia ) injury
Plasmid inducing with cDNA of rat telomerase reverse transcriptase (rTERT) and then pcDNA induce with sense sequence against were rTERT produced from jinsite biotechnology when plasmid pcDNA- rTERT induce into astrocytes by using lipofectamine 2000 reagent which is used as in-vitro method of induction of modified plasmid with astrocytes over proliferation of astrocytes controlled by mock plasmid which inserted with pcDNA- and rTERT astrocytes (Niccoli, 2011), after cell culture process resulting clone cells collected 400µg/ml G418 and maintained in Dulbecco’s modified Eagle’s medium supplemented as 100µg/ml G418 and fetal bovine serum, cultured astrocytes would be used for whether experiments (Niccoli, 2011).
Observing astrocytes proliferation in vitro
Modified clone astrocytes were selected from previous experimental methods and cultured in serum free Dulbecco’s modified Eagle’s medium then 10% fetal bovine and 600 ng/ml of 5-bromodeoxyuridine which are added into the media and incubate for 24 hours which going to be culturing (Niccoli, 2011) , serum contains of cell cycle regulator which can identify during western blot. During jinsite biotechnology cell cycle regulatory proteins either p15or p21 proteins were produced which could detected or sense by oilgodeoxynucelotides and phosphorothioate antisense (Niccoli, 2011). Cell cycle regulatory should be inhibited due to astrocyte proliferation by up regulation of TERT, inhibition of cell cycle regulatory proteins improves astrocyte proliferation. Astrocytes producing during proliferation neural growth factors such as transforming growth factor β1,glial cell line derived neurotrophin-3 (NT-3) which can detectable by western blot and enzyme-linked immunosorbent assays (Niccoli, 2011).
Inducing hypoxia and glucose deprivation to astrocytes
Culturing astrocytes with combined hypoxia and glucose deprivation (CHGD), before inducing with CHGD astrocytes should be incubated with 5-bromodeoxyuridine for 6 hours, CHGD induced astrocytes could be express NT-3 neuronal growth factor which can detectable in western blot and enzyme linked immunosorbent assays. Cell cultures of astrocytes performed in glucose free Dulbecco’s modified eagle’s medium incubated in humidified incubator under temperature of 37 and providing desire gases such as 93% of nitrogen gas, 5% of carbon dioxide, 2% of oxygen, associated with hypoxia and CHGD (Niccoli, 2011).
Modified astrocytes proliferation effect on neuronal survive on hypoxia
Astrocytes preserved in vivo condition like brain therefore which cultured in glucose free Dulbecco’s modified Eagles medium incubated by humidified incubator at 37℃ and providing appropriated gas distribution ( 93% of nitrogen gas, 5% of carbon dioxide, 2% of oxygen) when cultured CHGD astrocytes for 6 hours , fraction of times should be collect 10 ml of sample from the astrocyte which contains 6.4 × 106 cells per culture dish, sample collected as 12 hours per day or 24 hours per day , centrifuging samples to isolate supernatants which contains astrocytes and removing cell debris and then astrocytes were transferred into 3 culturing wells which has 4×105 neurons going to be incubate for 6 hours, during 6 hours of hypoxia neuronal apoptosis detected by using In Situ Cell Death Kit for terminal deoxynucleotidyltransferase mediated Dutp nick end labelling detection and apoptotic index calculation (Niccoli, 2011).
Astrocytes TERT expressed which detected by reverse transcriptase polymerase chain reaction which can detect TERT mRNA expression and western blot detecting TERT expression and immunocytochemistry used to detecting location of TERT IN hypoxia-ischemia conditioned brain, researchers find out results by described above methods such as TERT mRNA expressed on rat HI brain at 24 hours, neuronal TERT proteins like TERT/NeuN orTERT/glial fibrillary acidic protein expressed within 2 days in brain neurons and within 2,3days in astrocytes (Niccoli, 2011)
Neurotrophic factors expressed due to expression of TERT previously mentioned that growth factor b1, glial cell line derived neurotrophic factor, brain derived neurotrophic factor, NT-3 which were detected by western blot and enzyme linked immunosorbent assays (Niccoli, 2011), therefore come up with conclusion that TERT expression on astrocytes not changes capacity of astrocyte function such as synthesize glutamine eliminate glutamine and buffering the calcium.
NT-3 expressed due to TERT expression in astrocytes with CHGD condition, as observed was NT-3 expression identified earlier 2 hours and increasing until 24 hours of CHGD, TERT inhibited during increased NT-3 expression by using antisense oligonucleotides (Niccoli, 2011).
Proving the hypothesis of test should be considering about negative results therefore researchers undertaken astrocytic media with CHGD and astrocytic media without CHGD to analyse and comparing results because of TERT can up regulate the expression of neural growth factors such as NT-3 , researchers find out neuronal survival potential due to TERT expression levels of astrocytes using to detect by terminal deoxynucleotidyltransferase mediated Dutp nick end labelling staining test also detected neuronal apoptosis by TERT sense or antisense proteins in astrocyte cultured media, results obtained observed neuron apoptosis after 6 hours of hypoxia if neurons cultured with conditioned media without CHGD, neuron apoptosis decreased if neurons cultured in astrocyte conditioned media with CHGD treatment for 12 hours or 24 hours. (Niccoli, 2011) (Qu, 2011)
Plasmid producing TERT protein upregulate TERT activity or TERT antisense oligonucleotides inhibit TERT expression, but already mentioned that plasmids expressed TERT in astrocytes but not in neuron after transduction the astrocytes induced with HI brain neuron cells. Ki67 staining used to showing cellular proliferation marker found on TERT expression in astrocytes induced with HI injury (Niccoli, 2011). According to research studies determining the effect of TERT on HI brain, as measured infract volume of injured brain within 3,5, 7 days after HI condition using cresyl violet staining, identified that brain infract volume is decreasing on TERT expression while brain infract volume is decreasing on TERT antisense oligonucleotides. (Niccoli, 2011) (Qu, 2011)
Discussion and conclusion
Experimental studies about expression along with astrocytes proliferation researchers did the experiments from HI injured brain cells of mice inducing with controlled and modified astrocytes and modified astrocytes and observed results and compared as mentioned in previous methodology and results, main target of the experimental studies was possibility to provide therapeutic target to treat age related brain disorders by upregulating TERT to enhancing the self-renewal, therefore we have proof about telomere upregulation in mice by modifying single hallmark of aging, therapeutic target and interest of commercial enterprises to delaying or preventing age associated conditions although researches succeed to found that TERT function is enhance withp15 and NT-3 expression in astrocytes. As known telomerase enzyme/ TERT activity was producing telomere genes on chromosome during embryonic stage and maintaining the telomere length during each cell division but it also plays major role on regulation of calcium ions distribution, metabolism, neuronal growth factor production an apoptosis especially in the central nervous system through this case studies experimental studies about neurodegenerative disorders. TERT enhancing with the NT-3 expression already mentioned but prolong progressing expression of NT-3 had chance to cause neuroprotective effect by TERT against HI injury on rat brains therefore investigating to solve this problem. TERT expression in astrocytes also contributes cell reproduction on other cell types such as vascular endothelium and hepatocyte cells.
According to studies and experimental results TERT function improves axonal regeneration by inhibiting glial scar formed due to HI injury by inhibiting reactive astrocytes function such as forming local biochemical and physical barrier which can enhance with apoptosis to altering neuronal survival possibilities but here in this case neuronal apoptosis was inhibited due to earlier proliferating reactive astrocytes was inhibited that leads to inhibit molecules which were responsible neuronal damage.
According to review of all case studies about enhancing the telomere activity or preventing shortening telomere to preserving neuron cell neuronal survival or delaying neuronal apoptosis, concluded that as TERT expression in astrocytes with enhancement of p-15 cell cycle regulator and NT-3 expression can used as therapeutic target to treat age related neurodegenerative disorders but in this research they succeed to treating HI neonatal brain damage by used TERT expression as therapeutic target. Collectively not only the experimental studies that mentioned in this review article but also telomere gene therapy in animal models for treating aging or short telomere related diseases were succeed and developing therapeutic telomerase treatments in human aging associated diseases such as cancer, cardiovascular diseases (Oeseburg, 2010), as we know that aging is the major risk factor of many diseases therefore researchers tried and performing further more researches to improving the cellular and conserved cellular environment to older individual who have higher chance to getting aging related diseases that mentioned , tend to provide extend lifespan cell and improved broad health improvement like immortal the word we can used meaning like eligibility to survive against death or any other pathogenesis
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