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I'm looking at this diagram of homocysteine metabolism and see two distinct pathways that the amino acid may get metabolized to: with vitamin B12 it gets converted back into methionine, while with B6 it gets converted to cysteine.
I've followed the links for the products that Cysteine gets metabolized to and could not find any references to cognitive effects. Are there any cognitive effects of Cysteine or other amino acids that it gets metabolized to?
I'm interested in learning what are the possible effects of pharmacological (50mg - 100mg) doses of vitamin B6 on this metabolic cycle, while folate and B12 levels stay at the baseline levels. It seems to me that if vitamin B6 is more readily available, there will be a higher chance of homocysteine metabolized into Cysteine. Is this assumption correct? What are the implications to this metabolic cycle that may be caused by elevated levels of vitamin B6, while keeping folate and B12 at baseline levels?
I'm particularly interested in the implications of this process on dreaming. There has been numerous reports among dreamers that 50-100mg of vitamin B6, taken in the 3rd-5th sleep cycle produces very intense and vivid dreams, sometimes featuring spontaneous awareness (lucid dreams), while B-complex vitamins (B6, B12, Folate, Niacin, etc) appear to produce the opposite effect - very vague dream recall.
I'm trying to understand if there is indeed a connection between the metabolic pathway that homocysteine may take and the vividness of dreams, capacity to recall them clearly and the phenomenon of being able to spontaneously realize the fact that the person is dreaming while still asleep.
Thank you for your input!
Diagram obtained from here
Your shown homocysteine pathway is way too simple. Especially the production of the neuromodulator H2S from excess sulfur amino acids is not shown. In the last years many more enzymes and reactions have been discovered. I have summarized human sulfur amino acid metabolism in reactome.org, so please use this link to discover all the details and new papers. I'll also append some of the relevant papers below.
Brosnan, JT, Brosnan, ME The sulfur-containing amino acids: an overview 2006 J Nutr PMID 16702333
Remethylation of homocysteine to methionine can also happen using betaine as a methyl donor. This reaction is also part of choline catabolism.
Li, F, Feng, Q, Lee, C, Wang, S, Pelleymounter, LL, Moon, I, Eckloff, BW, Wieben, ED, Schaid, DJ, Yee, V, Weinshilboum, RM Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: common gene sequence variation and functional characterization 2008 Mol Genet Metab PMID 18457970
Bearden, SE, Beard RS, Jr, Pfau, JC Extracellular transsulfuration generates hydrogen sulfide from homocysteine and protects endothelium from redox stress 2010 Am J Physiol Heart Circ Physiol PMID 20817827
Dominy, JE, Stipanuk, MH New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant 2004 Nutr Rev PMID 15497768
Stipanuk, MH, Ueki, I Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur 2011 J Inherit Metab Dis PMID 20162368
Chiku, T, Padovani, D, Zhu, W, Singh, S, Vitvitsky, V, Banerjee, R H2S biogenesis by human cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia 2009 J Biol Chem PMID 19261609
How Do Various Cortisol Levels Impact Cognitive Functioning?
Children who grow up in stressful environments often have elevated levels of "the stress hormone” cortisol, which can impair cognitive development. New research shows that some children growing up with adversity actually have low levels of cortisol, which is also linked to compromised cognitive functioning.
A new study has identified how specific levels of cortisol affect the cognitive development of children who are growing up with the stress associated with family adversity, living in poverty, and/or homelessness.
The June 2015 study , “Tracing Differential Pathways of Risk: Associations Among Family Adversity, Cortisol, and Cognitive Functioning in Childhood,” was conducted at the University of Rochester, the University of Minnesota, and Mt. Hope Family Center, and published in the journal Child Development.
The statistics of childhood poverty are alarming. It’s estimated that four out of every ten American children currently live in low-income households, according to research from the National Center for Children in Poverty at Columbia University's Mailman School of Public Health.
The primary aim of the recent study was to gain a better understanding of how various cortisol levels impact a child’s social–emotional adjustment, as well as, his or her language, motor, and cognitive functions.
The researchers found that children with relatively high or relatively low levels of cortisol are more likely to experience learning deficits and cognitive delays.
Cortisol is generally referred to as “the stress hormone” because it’s secreted into the bloodstream in larger quantities during stressful situations or any environment that triggers the flight-or-fight response.
There are two kinds of stress: good stress (eustress) and bad stress (distress). Optimal levels of cortisol may be linked to eustress. In certain circumstances, the right amount of cortisol fuels your passion and gives you the oomph needed to seize the day.
It appears that low levels of cortisol could be a biomarker for depression, apathy, or hopelessness about someone's possible futures. Elevated levels of cortisol could be directly linked to the distress caused by environmental stressors.
Three Different Cortisol Profiles Impact Cognitive Functioning
For the new study, researchers measured children's cortisol levels when they were two, three, and four years old. When each child was two years old, the researchers observed them playing with their mothers and collected extensive information about family dynamics, such as how stable the family home appeared to be and whether children had been exposed to domestic violence.
When the children were four years old, researchers measured their cognitive abilities. The study found that children with relatively higher and lower cortisol profiles had reduced levels of cognitive functioning at age four.
In a press release, Jennifer H. Suor, doctoral student in clinical psychology at the University of Rochester and the study's first author explained the findings saying,
Overall, we found three cortisol profiles among the children, which were categorized as elevated, moderate, and low. We found that children's cortisol levels remained relatively stable across the three years. And we discovered that exposure to specific forms of family adversity when children were two years old predicted their cortisol profile, which in turn was linked with notable differences in children's cognitive functioning at age four.
The study reported that about 30 percent of the children in the study maintained relatively higher cortisol levels 40 percent of the children maintained lower cortisol levels and the remaining 30 percent maintained moderate cortisol levels. Interestingly, children on either end of the spectrum of cortisol levels had experienced some type of family instability.
Children with the highest cortisol levels had experienced harsher and more traumatic interactions with a caregiver or parent. On the flip side, children with moderate cortisol levels were exposed to relatively less family adversity at age two and also had the highest cognitive abilities at age four.
The researchers are unsure of the exact mechansims that link cortisol levels and cognitive functioning. They hypothesize that too much cortisol may have toxic effects on parts of the brain that are important for cognitive functioning. Too little cortisol might impede the body's ability to recruit the biological resources necessary for optimal cognitive development.
What Is N-Acetyl Cysteine?
N-acetyl cysteine (or NAC for short) is a precursor to the sulfur-rich amino acid L-cysteine.
L-cysteine is found naturally in many protein– and sulfur-rich foods, including meat, poultry, egg yolks, broccoli, Brussels sprouts, garlic, oats, onions, and red bell peppers.
L-cysteine is classified as a “conditionally essential” amino acid, which means that under certain conditions, we need to consume extra sources because our body cannot make adequate amounts of it from other amino acids.
NAC is not an essential nutrient and it’s not found in nature, so there isn’t an established daily requirement for N-acetyl cysteine.
NAC may be most familiar to physicians since it’s used as a standard treatment for acetaminophen toxicity (overdose of the active ingredient in Tylenol®). NAC is also used medically as an aerosolized mist to break up mucus in the airways of patients with cystic fibrosis.
It’s also a common dietary supplement ingredient that has more functions than simply being a protein building block. Let’s explore.
When taken by mouth: N-acetyl cysteine is LIKELY SAFE for most adults. It can cause nausea, vomiting, and diarrhea or constipation. Rarely, it can cause rashes, fever, headache, drowsiness, low blood pressure, and liver problems. N-acetyl cysteine has an unpleasant odor that may make it hard to take.
When given by IV: N-acetyl cysteine is LIKELY SAFE for most adults, when given by IV as a prescription medication. Rarely, it can cause rashes, fever, headache, drowsiness, low blood pressure, and liver problems.
When inhaled: N-acetyl cysteine is LIKELY SAFE for most adults, when used as a prescription medication. When inhaled (breathed into the lungs), it can also cause swelling in the mouth, runny nose, drowsiness, clamminess, and chest tightness.
Cognitive effects of adjunctive N -acetyl cysteine in psychosis
Cognitive deficits are predictors of functional outcome in patients with psychosis. While conventional antipsychotics are relatively effective on positive symptoms, their impact on negative and cognitive symptoms is limited. Recent studies have established a link between oxidative stress and neurocognitive deficits in psychosis. N -acetylcysteine (NAC), a glutathione precursor with glutamatergic properties, has shown efficacy on negative symptoms and functioning in patients with schizophrenia and bipolar disorder, respectively. However, there are few evidence-based approaches for managing cognitive impairment in psychosis. The present study aims to examine the cognitive effects of adjunctive NAC treatment in a pooled subgroup of participants with psychosis who completed neuropsychological assessment in two trials of both schizophrenia and bipolar disorder.
A sample of 58 participants were randomized in a double fashion to receive 2 g/day of NAC ( n = 27) or placebo ( n = 31) for 24 weeks. Attention, working memory and executive function domains were assessed. Differences between cognitive performance at baseline and end point were examined using Wilcoxon's test. The Mann–Whitney test was used to examine the differences between the NAC and placebo groups at the end point.
Participants treated with NAC had significantly higher working memory performance at week 24 compared with placebo ( U = 98.5, p = 0.027).
NAC may have an impact on cognitive performance in psychosis, as a significant improvement in working memory was observed in the NAC-treated group compared with placebo however, these preliminary data require replication. Glutamatergic compounds such as NAC may constitute a step towards the development of useful therapies for cognitive impairment in psychosis.
Biological and psychological effects of PE could be partly explained through epigenetic mechanisms. The term 𠇎pigenetics,” coined by Waddington (1939), is based on a conceptual model designed to account for how genes might interact with their environment to produce the phenotype (Waddington, 1939 Fernandes et al., 2017).
In particular, epigenetics is referred to all those mechanisms, including functional modifications of the genome such as DNA methylation, post-translational histone modifications (i.e., acetylation and methylation) and microRNA expression (Deibel et al., 2015 Grazioli et al., 2017), which tend to regulate gene expression, modeling the chromatin structure but maintaining the nucleotide sequence of DNA unchanged.
The current literature clearly demonstrates that these mechanisms are strongly influenced by different biological and environmental factors, such as PE (Grazioli et al., 2017), which determine the nature and the mode of epigenetic mechanisms activation.
Epigenetics plays an essential role in neural reorganization, including those that govern the brain plasticity (Deibel et al., 2015). For example, a growing body of evidence indicates that regulates neuroplasticity and memory processes (Ieraci et al., 2015).
Several animal studies reveal how motor activity is able to improve cognitive performances acting on epigenetic mechanisms and influencing the expression of those genes involved in neuroplasticity (Fernandes et al., 2017). The main molecular processes that underlie the epigenetic mechanisms are the following: through DNA methylation, histone modifications and microRNA expression (Fernandes et al., 2017).
DNA methylation is a chemical covalent modification on the cytosine of the double stranded DNA molecule. It has been recognized that DNA methylation plays a key role in long-term memory (Deibel et al., 2015 Kim and Kaang, 2017). In particular, mechanisms related to DNA methylation relieve the repressive effects of memory-suppressor genes to favor the expression of plasticity-promoting and memory consolidation genes. Several evidences showed that PE is able to coordinate the action of the genes involved in synaptic plasticity that regulate memory consolidation (Molteni et al., 2002 Ding et al., 2006).
Histone modifications are post-translational chemical changes in histone proteins. They include histone methylation/demethylation, acetylation/deacetylation, and phosphorylation, all due to the activity of specific enzymes, which modify the chromatin structure, thereby regulating gene expression. It has been demonstrated that histone acetylation is a requisite for long-term memory (LTM) (Barrett and Wood, 2008 Fernandes et al., 2017). In animals, motor activity increases these genetic mechanisms in the hippocampus and the frontal cortex, improving memory performances in behavioral tasks (Cechinel et al., 2016). Recently, following 4 weeks of motor exercise, it has been evidenced an increasing of the activity of enzymes involved in histone acetylation/deacetylation, the epigenetic mechanisms that determine an enhancing in the expression of BDNF (Maejima et al., 2018).
MicroRNAs (miRNAs) are small, single stranded RNA molecules able to inhibit the expression of target genes. They are widely expressed in the brain, participating in epigenetic mechanisms and acting as regulators of numerous biological processes in the brain, ranging from cell proliferation, differentiation, apoptosis, synaptic plasticity, and memory consolidation (Saab and Mansuy, 2014). Recent evidences demonstrate that PE can mitigate the harmful effects of traumatic brain injury and aging on cognitive function by regulating the hippocampal expression of miR21 (Hu et al., 2015) and miR-34a (Kou et al., 2017). Furthermore, PE contributes to attenuate the effects of stress-related increase in miR-124, involved in neurogenesis and memory formation (Pan-Vazquez et al., 2015).
4 CYSTEINE AND HOMOCYSTEINE AS BIOMARKER OF VARIOUS DISEASES
4.1 Cardiovascular diseases
In developing and developed countries, cardiovascular disease (CVD) is the leading cause of death worldwide. Many studies relate to elevated Hcy role in the development of various forms of vascular diseases (Hannibal & Blom, 2017 ). According to some researchers, the concentration of plasma Hcy above 10 µmol/L is associated as a risk factor in the development of CVD and ischemic heart disease (Ostrakhovitch & Tab ibzadeh, 2019 ). A meta-analysis indicated that a 25% elevated plasma Hcy level was associated with 10% and 20% increased risk of CVD and stroke, respectively. Another meta-analysis showed that when serum Hcy level decreased by 3µmol/L, a 16% reduction in coronary heart disease was seen. A 5 µmol/L increase in plasma increased the relative risk of coronary heart disease by 1.6–1.8 times (Ntaios, 2015 ). Another meta-analysis showed that for each 5 μmol/L increase in Hcy, the risk of mortality increased by 32%, and the risk of heart disease increased by 52% (Shiao, Lie, & Yu, 2018 ). Hcy may cause CVD through various mechanisms such as the increased proliferation of muscle cells that cause narrowing of vessels, alter blood coagulant properties, cause oxidant injury to the vascular endothelium, and damage arterial walls (Mishra, 2016 ). Table 4 summarizes the effects of hyper Hcy on cardiac health.
Children with parental history of
CVD had higher serum Hcy levels than those without such history
Hcy was positively associated with both diastolic and systolic blood pressures for instance, when Hcy concentration increased by 5 μmol/L, diastolic and systolic blood pressure in men increased by 0.5 and 0.7 mmHg, respectively, and in case of women, Hcy and blood pressure showed stronger correlation and increased by 0.7 and 1.2 mmHg (diastolic and systolic) (Ganguly & Alam, 2015 ).
Epidemiological research demonstrated a U-shaped relationship between cardiovascular diseases and tCys after adjustment of other risk factors and Hcy (De Chiara et al., 2012 ). Van den Branbdhof and his colleagues found no relationship between tCys and the risk of coronary heart disease. A study was conducted to investigate the relationship between tCys and risk of mortality and CVD hospitalizations on men and women in the Hordaland Hcy Study cohort and concluded that tCys was not associated with all cause of CVD or non-CVD mortality (van den Brandhof, Haks, Schouten, & Verhoef, 2001 ). A study was conducted, later on, to determine the relationship between tHcy and CVD and non-CVD-related mortality and morbidity, and results demonstrated that tHcy as a biomarker of CVD and non-CVD mortality and morbidity (El-Khairy, Vollset, Refsum, & Ueland, 2003 ).
4.2 Ischemic stroke
In order to determine the association between elevated tHcy level in acute stroke with mortality, an experiment was conducted to analyze whether during the convalescent phase after an acute ischemic stroke, change in Hcy level in plasma contributed to the risk of ischemic stroke. The study showed that during the convalescent phase, elevated Hcy was independently associated with an increased risk of recurrent ischemic stroke after the index cerebrovascular event. So, researcher concluded that tHcy used as the biomarker of ischemic stroke during the convalescent phase of stroke (Shi et al., 2018 ). Another study showed that 5 µmol/L increase in tHcy elevates the relative risk of ischemic stroke by 1.5 times. When serum Hcy decreased 3µmol/L a 24% reduction in ischemic stroke was seen (Ntaios, 2015 ).
4.3 Neurological disorders
Hcy is transported in the brain which has a limited capacity for Hcy metabolism. Brain tissues have different mechanisms to lower down and maintain Hcy level such as efficient recycling through vitamin B12-dependent methionine synthase (only enzyme in the brain involved in Hcy conversion into methionine), catabolism through cystathionine beta-synthase to cystathionine (a non-noxious product which converts further into cysteine), and export to external circulation (Ganguly & Alam, 2015 ).
In the brain, accumulation of Hcy was associated with increased tHcy and SAM in cerebrospinal fluid. Induced Hcy caused endothelial and astrocytic dysfunction which resulted in altered neuronal function. Elevated levels of Hcy led to an enhanced excitatory glutamatergic neurotransmission in different brain regions which caused neuronal damage. In short, Hcy caused redox imbalance and increased oxidative stress and production of free radicals in many cells including endothelial, glial, and neuronal cells and led to several neurological disorders (Lehotský et al., 2016 ). On the other hand, cysteine plays an important role in redox homeostasis. Redox-modulated events play important roles not only in peripheral tissues but also in the brain where cysteine disposition is central to these pathways. Disrupted redox homeostasis played an important role in disease progression and neurodegenerative disorders, and Cys, being an antioxidant, is responsible for neutralizing much of the oxidative damage generated (Paul et al., 2018 ).
4.4 Autism spectrum disorder
Nowadays autism spectrum disorder (ASD) is the topic of many types of research with AAs being used as a biomarker of ASD. ASD is the neurodevelopmental disorder that occurs early in life and is associated with abnormal functioning of CNS. ASD is estimated to be prevalent in almost 11.3 per 1,000 children age 8 years. Besides cysteine and Hcy, other AAs like tyrosine, threonine, methionine, phenylalanine, and other brain AAs also considered as a biomarker of ASD (Żurawicz & Kałużna-Czaplińska, 2015 ). Studies also reported less concentration of Cys in ASD patients and control after overnight fasting. These findings are compatible with increased oxidative stress and decreased detoxification capacity in ASD (ElBaz, Zaki, Youssef, ElDorry, & Elalfy, 2014 Geier et al., 2009 ). Similarly, another study reported low intracellular cysteine concentration in children suffering from ASD as compared to the healthy group and observed elevated levels of Hcy in the peripheral blood mononuclear cells (Suh, Walsh, McGinnis, Lewis, & Ames, 2008 ). Tu, Chen, and He, ( 2012 ) observed a high concentration of Hcy in the fasting plasma of ASD patients. Moreover, a high level of Hcy in plasma and urine may due to vitamin B deficiency. James et al. ( 2004 ) observed a low level of Hcy in fasting plasma, this difference may due to different ages of participants, and James et al also observed low level of cysteine in the plasma of ASD children as compared to the control group. There are two studies by Kałużna-Czaplińska, Żurawicz, Struck, and Markuszewski ( 2014 ), and Kałużna-Czaplińska, Michalska, and Rynkowski ( 2011 ) also reported a high level of Hcy in the urine of ASD children than the healthy group.
Dementia is not a specific disease but a clinical syndrome that includes different diseases like Parkinson's disease, Alzheimer's disease (AD), Lewy body diseases (LBD), and vascular dementia (VAD). A 50% increase in the risk of dementia was seen when 5-µmol/L increase was observed in tHcy. Hcy may be harmful to nervous tissue due to various reasons since it causes increased stimulation of receptors, increased activation of pathological protein formation, and direct neurotoxicity (Sławek & Białecka, 2015 ).
4.6 Alzheimer's disease
The most common form of dementia is AD, and the risk of AD is almost double in people over 50 years of age with tHcy level higher than 14 μmol/L (Sławek & Białecka, 2015 ). The abnormal increase in tHcy can be used as a biomarker of AD. HHcy in AD may be due to folate or vitamin B12 deficiency. A study is conducted to explore the relationship between HHcy and vitamins as the disease progresses (Farina, Jernerén, Turner, Hart, & Tab et, 2017 ). A meta-analysis showed that high tHcy and low folate and vitamin B12 concentrations may act as a risk factor of AD (Shen & Ji, 2015 ). Disrupted cysteine metabolism is seen in AD patients with some studies reporting an increased level of Cys in the AD patients. This could be attributed to impaired uptake of Cys into the cells, decreased level of H2S and disruption of Cys neuronal transporter in AD (Paul et al., 2018 ).
For epilepsy (sudden occurrence of seizures in behavior), peripheral biomarkers are absent notably, but in humans, few studies suggested these biomarkers. Redox changes were seen in animal models of epilepsies, and cysteine was considered as a biomarker of oxidative damage. The ratio of Cys/Cys (cysteine/cysteine) has been proposed to serve as a biomarker of redox status in plasma and tissues in humans and in animals. A study on rats concluded that a decrease in Cys and ratio of Cys/Cys in plasma could act as a redox biomarker in temporal lobe epilepsy (Liang & Patel, 2016 ). Hcy and its metabolites homocysteine thiolactone (HT) causes seizures by different unknown mechanisms. Elevated tHcy and HT levels have been identified as risk factors for the development of numerous brain disorders including epilepsy (Hrnčić et al., 2014 ).
4.8 Head injury
Head injury is among the numerous causes of mortality and morbidity in young people. Total plasma homocysteine is used as a biomarker of various neurological outcomes after head injury. An experiment showed that after a head injury, elevated tHcy has an independent impact on neurological outcomes. The study included patients (both males and females) who were admitted within 24 hr after head injury for 3 months, and their biochemical tests showed higher tHcy levels in severely injured patients and low tHcy in mildly injured patients. The elevated levels of tHcy after a head injury can be due to metabolic stress response similar to the results of subarachnoid hemorrhage (a type of stroke), caused by down regulation of cystathionine-β-synthase. The result showed a significant relationship between tHcy and neurological outcomes (Dhandapani et al., 2018 ).
Diabetes is the most common metabolic disorder associated with hyperglycemia. It is suggested that a high level of Hcy in diabetes is the biomarker of microvascular complications like diabetic neuropathy, retinopathy, and nephropathy. Various studies also suggest that in poorly controlled type-2 diabetes mellitus increased level of Hcy is associated with increased risk of macrovascular complications like atherosclerosis and cardiovascular disease (Mundu, Kumar, Mitra, Kumar, & Sinha, 2017 ). HHcy in type 2 diabetes is associated with a high prevalence of nephropathy (Rujaswini, Praveen, Chowdary, Aanandhi, & Shanmugasundaram, 2018 ). Emerging evidence shows a positive role of L-cysteine-rich diet in several pathological conditions of type-2 diabetes and neurodegenerative diseases (Yin et al., 2016 ). Different studies showed that in diabetic patients, cysteine homeostasis changed and resulted in lower cysteine blood levels. In type 2 diabetic patients, L-cysteine (LC) supplementation lowered oxidative stress which was considered as a risk factor in the progression of vascular inflammation in diabetes (Manna & Jain, 2013 ). L-cysteine can be strongly considered in antidiabetic drugs in the management of type 2 diabetes (Salman, Refaat, Selima, El Sarha, & Ismail, 2013 ).
4.10 Diabetic neuropathy
Diabetic neuropathy is the condition of diabetes in which nerve damage and dysfunction occur. The study indicated that Hcy is independently associated with the prevalence of diabetic neuropathy in collective type 2 diabetic patients (Rujaswini et al., 2018 ). Research is conducted to finding out the relationship between elevated Hcy level and microvascular complications in diabetics, and results showed that serum Hcy level in diabetes patients with microvascular complications is an important biomarker (Mundu et al., 2017 ).
4.11 Gestational diabetes mellitus
Gestational diabetes mellitus (GDM) is a metabolic complication with glucose intolerance during pregnancy. High plasma level of Hcy was found in pregnant women with GDM as compared to nonpregnant women with normal glucose tolerance (Gong et al., 2016 ). (Tarim et al., 2004 ) conducted a study to determine whether elevated plasma Hcy is associated with GDM in Turkish women and found that women with gestational diabetes with abnormal screening test results but normal oral glucose tolerance test results have higher Hcy levels than normal pregnant women. A cross-sectional study was conducted, which include 223 pregnant women between 24 and 28 weeks of gestation. Sample divided into 3 groups based on their glucose tolerance, glucose intolerance, gestational diabetes, and normal controls, and serum Hcy level was prospectively measured. The result found an elevated level of Hcy during the second trimester among women with gestational diabetes mellitus as compared to normal controls (Guven, Kilinc, Batukan, Ekerbicer, & Aksu, 2006 ).
4.12 Renal dysfunction
Mild-to-moderate hyperhomocysteinemia is often associated with impaired kidney function. The majority of renal disease patients reported HHcy (Barroso et al., 2017 ). The kidney is the major site of Hcy metabolism, and any disturbance in Hcy metabolism pathways leads to an abnormal increase in its level which causes many renal problems and impairment in renal function. Renal dysfunction is responsible for further accumulation of Hcy, thus causing chronic renal failure. Studies showed that Hcy toxicity caused ischemic–reperfusion which led to kidney damage. Imbalanced homeostasis and increased oxidative stress and reactive oxygen species (ROS) which caused glomerular endothelial dysfunction also resulted in a change in glomerular filtration rate, which induced renal dysfunction as shown by increased Hcy and Cys. And Cys-albumin increased in end-stage kidney disease (Ostrakhovitch & Tabibzadeh, 2015 ). A study concluded that in nephropathic cystinosis, N-acetyl-cysteine (NAC) reduced oxidative stress and improved renal function (de Faria Guimaraes et al., 2014 ). Some recent studies demonstrated that NAC might prevent contrast nephropathy (impairment of renal function). It has anti-inflammatory and antioxidant effects that could prevent renal dysfunction (Aldemir et al., 2016 ).
4.13 Renal cell carcinoma
In the United States almost 13,000 deaths and 50,000 new cases so that, RCC detection is increasing day by day. Kidney plays an important role in the regulation of plasma AAs and in the clearance of nitrogenous substances from our bodies. From glomerular filtration, renal tubule epithelial cells reabsorb AAs so that most tumors arise from renal tubule cells, and due to these tumors, change in the reabsorption of AAs is seen. In a study, a researcher obtained serum of 189 patients suffering from RCC at Fox Chase Cancer Center and stored at −70C. Serum samples of control groups from different sources like employees, spouses of patients, and other individuals at Fox Chase Cancer Center were collected. With the help of amino acid analyzer, their sample comparison showed alternations between 15 out of 26 AAs like taurine, threonine, glycine, alanine, serine, asparagine, glutamate, citrulline, methionine, tyrosine, ornithine, histidine, phenylalanine, and proline were decreased in RCC patients while cysteine and arginine were increased. This study shows that a significant difference between serum AAs may use for screening tests of RCC and may have potential clinical uses in the determination of RCC (Mustafa et al., 2011 ).
Murphy et al. ( 2011 ) conducted a study that showed that high concentrations of cysteine were linked with low risk of cell carcinomas and adenocarcinomas. DNA methylation is one of the risk factors of cancer, and one-carbon metabolism (OCM) played an important role in providing a methyl group. OCM involved many factors like vitamin B12, Hcy, folate, vitamin B6, and methionine. Decrease and accumulation of even one substance affected DNA methylation, genomic material, and tumor suppressor genes which lead to malignant transformation. In another study, the analysis showed elevated Hcy contributed to cancer (Yang, Li, Deng, & Wang, 2018 ).
Zhang, Wen, Wu, Guo, and Cui ( 2015 ) performed a meta-analysis to examine the relationship between Hcy and folate with cancer and concluded that folate deficiency and increased serum Hcy associated with overall risk of cancer. Ovarian cancer also has a connection to the level of Hcy. Cancer cells derived from ovarian cancer patients had a deficit in the ability to remethylate Hcy. This changed metabolic condition results in elevated levels of serum Hcy (Kim, Kim, Roh, & Kwon, 2018 ).
4.15 Digestive tract cancer
Digestive tract cancer is the malignant gastrointestinal disease that covers gastric, esophageal, and colorectal cancer. Digestive tract cancer constitutes 22% deaths due to cancer every year, and malignancies of these cancers may be linked to hyper-methylation and hypo-methylation. If we find out the substance that affects cellular methylation, then digestive tract cancer diagnoses in early stage and several lives can be safe. Studies showed that an increase in Hcy caused elevation in S-adenosylhomocysteine (SAH) which is a by-product of remethylation reaction. Elevation in SAH results in hypo-methylation of lymphocyte DNA. So, an indirectly elevated level of Hcy affects cellular methylation. Dose–response analysis also showed that 5 μmol/L increase in the concentration of Hcy enhanced 7% risk of digestive cancer (Xu et al., 2018 ). Several studies linked Hcy relation with digestive tract cancer like, and Wang et al. ( 2007 ) observed that people having a high concentration of Hcy than those having low showed an increased occurrence of gastric cancer means elevated Hcy increased risk of digestive tract cancer. One meta-analysis demonstrated the relationship between blood tHcy and prostate cancer (Collin et al., 2010 ). Another meta-analysis found that higher tHcy levels increased gastric cancer risk (Xu, Cheng, & Zhu, 2016 ).
4.16 Lung cancer
Lung cancer (LC) is also common and fatal cancer in the world. A meta-analysis showed that serum Hcy was higher in LC patients than the serum level of Hcy in the control group. Further study demonstrated the difference of serum Hcy level in normal and LC patients while vitamin B6, folate level observed low than normal in these patients. This analysis showed that in OCM, vitamin B6 and folate act as a protective factor while Hcy contributes to lung cancer and can be used as a biomarker (Yang et al., 2018 ).
4.17 Colorectal cancer
In the United States, colorectal cancer (CRC) is the 3rd most commonly diagnosed cancer and the leading cause of death in both males and females. Many meta-analyses were focused on determining the effects of dietary folate, supplements, and fiber on cancer and indicated that increased CRC risk is associated with increased tHcy and decreased folate level. The reason is that HHcy is responsible for oxidative stress and induction of the inflammatory responses, thus increasing the risk of CRC (Shiao et al., 2018 ). A study was conducted with postmenopausal women to assess the association between cysteine and Hcy with the incidence of CRC, and the result indicated that elevated plasma Hcy was associated with increased risk of CRC, whereas high cysteine level was associated with decreased risk. So HHcy low cysteine is risk factors for incident CRC (Miller et al., 2013 ). Patients with the inflammatory intestinal disease have a greater chance of developing colorectal cancer when their Hcy levels are higher than in healthy individuals (Keshteli, Baracos, & Madsen, 2015 ). Dietary factors and lifestyle may contribute to the increasing CRC incidence like obesity, fat diet, alcohol consumption, and smoking (Baena & Salinas, 2015 ). Fat diet is also considered as the main cause of CRC the reason is maybe that the high-fat diet is responsible for increasing plasma Hcy levels in our body (Jakubowski, 2019 ).
Hyperhomocysteinemia has been reported with vitiligo (multifactorial pigmentary disorder). Worldwide, the prevalence of this disease is around 1%. Its etiology may be due to the destruction of melanocytes, oxidative stress, and autoimmunity. Many studies have reported an elevated level of Hcy in vitiligo and concluded Hcy increased oxidative stress and disrupted melanocytes. A meta-analysis reported a high level of Hcy and low levels of folate, vitamin B12, and vitamin B6 in the patients suffering from vitiligo as compared to the control group. Several hypotheses like inhibition of melanin synthesis enzyme (tyrosinase) or increased ROS production by Hcy can explain the elevated level of Hcy in vitiligo (Tsai, Kuo, & Huang, 2018 ).
4.19 Homocysteine and body composition
A study conducted on nine homocystinuria patients showed that reduced fat mass is common in these patients and alternation in choline and Hcy pathways affects lipid metabolism and body mass (Poloni et al., 2014 ). Yin et al. ( 2016 ) investigated the effect of L-cysteine and found that L-cysteine effectively reduces final body weight, body weight gain, food intake, and feed efficiency in rats.
Results and discussion
The three groups did not differ in years in education, MMSE or pre-intervention physical activity level, F (2, 97) = .77, p = .466, F (2, 97) = 1.93, p = .151, F (2, 97) = .98, p = .378, respectively (see Table 2). PASE did not differ significantly between groups after the intervention either, t(98) = 1.547, p = .125 (control M = 35.62, SD = 21.93, cyclist M = 44.47, SD = 26.09). 72% of participants completed the trial during the warmer months to maximise adherence to the trial. Furthermore, E-bike cyclists (M = 1.86, SD = 1.76), pedal cyclists (M = 2.22, SD = 1.76) and non-cycling control participants (M = 1.92, SD = 1.65) did not differ in the frequency of doing these other activities, H (2, 97) = 1.08, p = .582, as reported in the PASE. E-bike cyclists (M = 1.53, SD = 1.41), pedal cyclists (M = 1.58, SD = 1.25) and non-cycling control participants (M = 1.62, SD = 1.65) also did not differ in the number of other activities they participated in, F (2, 97) = .033, p = .968. Finally, E-bike cyclists (M = 2.50, SD = 2.47), pedal cyclists (M = 2.58, SD = 2.39) and control participants (M = 2.46, SD = 2.32) did not differ in the time spent completing these activities, H (2, 97) = .049, p = .976, as reported in the PASE. Participants continued to report their other physical activities (additional to cycling) that they conducted throughout the cycling trial (complete diaries received, N = 81). Again, the average time spent completing other activities did not differ (E-bike [M = 1.21, SD = .54], pedal [M = 1.21, SD = .38] and control participants [M = 1.27, SD = .30]) across the participant groups, F (2, 78) = .141, p = .869).
Cycling statistics during the trial
Participants kept a diary of their cycling activity during the trial and recorded the duration of each journey. We found e-bike cyclists spent marginally more time cycling on average each week than pedal cyclists, t(72) = 1.80, p = .076 (see Table 3 for Means and SDs). This is likely due to the ease associated with cycling with a motor, enabling the e-bike participants to cycle for longer periods of time. This indicates that e-bikes, due to supporting the cycling, may enable increased activity and durations of cycle rides. Many of the participants commented that they felt they could go further on the e-bike as they could rely on it to get home if they could not manage it by themselves (see  for a qualitative account of factors affecting cycling behavior in ‘Older people’s microadventures outdoors on (e-) bikes’).
Additionally, e-bikers spent on average 26% of the time in the highest motor setting (turbo SD = 34), 7% in the next highest setting (sport, SD = 11), 24% in tour (SD = 22), 28% in eco (SD = 26), and 15% (SD = 26) with the motor off. This means that on average for only 15% of their cycling time, e-bike participants were not using the motor to aid their cycling, thus being comparable to pedal cyclists.
Participants (pseudonym) often included comments in their daily diary of cycling experience summing up their belief of the contribution to psychological well-being, for example:
"On Sunday I took the (E-)bike out for the afternoon to cheer myself up. Gloomy day but the countryside around is lovely so felt better when I came back!" (Alysia)
“After a stressful morning I had time to unwind on the (E-)bike.” (Christopher)
Executive function measures
Large effect sizes for improvement in executive function after exercise have been demonstrated  and we predicted an increase in executive function in the cycling groups, thus would expect significant interactions between group and session, with the control group not improving after the intervention. The ANOVA demonstrated that there was no interaction for verbal fluency (F(2, 97) = .739, p = .480), plus-minus task (F(2, 97) = 4.07, p = .667), letter updating (F(2, 97) = 1.92, p = .152), Eriksen interference score (F(2, 97) = .623, p = .538), all measuring executive function.
There was, however, a significant Group x Session interaction for the Stroop Interference score, a measure of inhibition, F(2, 97) = 3.77, p = .026, ƞ 2 = .072. The smaller the score, the less interference that the participant experienced from the written word being incongruent with the ink colour that they were reporting. This measure demonstrated improvement in both the cycling groups after the intervention, for e-bike cyclists, t(37) = 2.75, p = .009 , and for pedal cyclists, t(35) = 5.30, p = .000, with less interference after the intervention (see Fig 1), which was not the case for non-cycling control participants, t(25) = .03, p = .974.
There was also a significant interaction between session and group for Go RTs in the Stop-It task, a measure of processing speed, F(2, 97) = 3.78, p = .026, ƞ 2 = .072 (see Fig 2). E-bike cyclists had marginally faster RTs after the intervention compared to baseline, t(37) = 1.97, p = .056, whereas pedal cyclists did not, t(35) = .87, p = .391, and the non-cycling controls had a trend towards slower RTs, t(25) = -1.75, p = .092.
There was no overall difference between the groups for verbal fluency scores (F(2, 97) = 2.18, p = .119), the plus-minus task (F(2, 97) = .368, p = .693), Stroop interference scores (F(2, 97) = 1.00, p = .905), the Eriksen flanker task (F(2, 97) = 2.28, p = .107) or Stop-IT go-RTs, measuring processing speed (F(2, 97) = .930, p = .398). There was a main effect of group for the letter updating task, F(2, 97) = 4.20, p = .018, with both cycling groups being higher overall in accuracy than the non-cycling control group, t(62) = 2.44, p = .017 (e-bike cyclists compared to non-cycling controls), t(60) = 2.54, p = .014 (pedal cyclists compared to non-cycling controls). As there were no significant differences in baseline performance across groups (see S1 File), this main effect is mainly driven by the after intervention accuracy being higher for the cycling groups, t(98) = 3.72, p = .001 (see Fig 3) as demonstrated by the interaction.
There was no session effect for the plus-minus task (F(2, 97) = 2.81, p = .097) or for the go RTs in the Stop-IT task, F(2, 97) = .193, p = .662. There was a session effect for the groups overall, with improvement after the 8-week period for verbal fluency (F(2, 97) = 8.50, p = .004, ƞ 2 = .081), letter updating, F(2, 97) = 27.41, p = .000, ƞ 2 = .221 (see Table 4 for all Means and SDs for the executive function measures), the Stroop interference score (mainly driven by the improvement in the cycling groups after the intervention, as well as practice effects improving performance after the intervention), F(2, 97) = 15.96, p = .000, ƞ 2 = .141, and a marginal session effect of the reduction of interference in the Eriksen flanker task, F(2, 97) = 3.73, p = .056, ƞ 2 = .037. These session effects alone are likely due to performance improving overall after the intervention as a result of practice.
There was no group, session or interaction effect on the MMSE scores, group effect F(2, 97) = 1.35, p = .264, session effect F(2, 97) = .328, p = .568, interaction F(2, 97) = .616, p = .542. The CERAD composite did not show an effect of cycling on recall either, group effect F(2, 97) = .874, p = .420, interaction F(2, 97) = .495, p = .611, which is not surprising given the lower effect sizes reported for exercise interventions on memory. There was a session effect for the CERAD composite score, F(2, 97) = 30.84, p = .000, reflecting better performance after the intervention, t(99) = -5.54, p = .000, ƞ 2 = .241, likely due to practice effects.
Spatial function measures
Despite evidence to suggest there are medium effect sizes for spatial function improvement after exercise  and we predicted an increase in this ability in the cycling groups, the ANOVA demonstrated that there was no interaction or group effect for the Mental Rotation Task Accuracy, group effect F(2, 97) = .874, p = .420, interaction F(2, 97) = .874, p = .420, Maze Errors, group effect F(2, 97) = .874, p = .420, interaction F(2, 97) = .874, p = .420, and Spatial Function Time (Composite of the completion times for the mazes and mental rotation task), group effect F(2, 97) = .874, p = .420, interaction F(2, 97) = .874, p = .420. Again, there was a significant session effect in the Spatial Function Time composite indicating the influence of practice on increased speed from completing the tests again after the intervention, F(2, 97) = 10.62, p = .002, ƞ 2 = .099.
Well-being and mental health questionnaires
As with spatial function and some of the executive function measures, we predicted to see an increase in well-being in the cycling groups compared to controls. The ANOVA demonstrated that there was no interaction, or group effect for the PWB, group effect F(2, 97) = .441, p = .644, interaction F(2, 97) = 1.48, p = .232, session effect, F(2, 97) = 1.95, p = .166, the SL, group effect F(2, 97) = 1.03, p = .363, interaction F(2, 97) = .340, p = .713, PANAS positive, group effect F(2, 97) = 1.95, p = .148, interaction F(2, 97) = 1.01, p = .370 or PANAS negative, group effect F(2, 97) = 1.52, p = .223, interaction F(2, 97) = 1.22, p = .300, and session F(2, 97) = 1.09, p = .742, (see Table 5 for Ms and SDs for all well-being measures). There was a session effect for Positive PANAS items, demonstrating an increase in the positive score in all groups after the intervention period, F (2, 97) = 8.92, p = .004, ƞ 2 = .072. This was also the case for the SL, F(2, 97) = 8.32, p = .005, ƞ 2 = .079.
There was also a marginal interaction between session and group for the mental health component of the SF-36, F(2, 97) = 4.25, p = .017, ƞ 2 = .081, with the e-bike cyclists increasing in this score, t(37) = -3.45, p = .001, but pedal cyclists and non-cycling controls not, t(35) = 1.56, p = .128, t (25) = 1.03, p = .311 (see Fig 4). There was also a significant session effect (with an increase in their mental health score after the intervention period), F(2, 97) = 5.13, p = .026, ƞ 2 = .050, but no group effect, p > .05, F = .78. This interaction was not the case for the physical health component of this measure but there was a session effect, F(2, 97) = 7.74, p = .007, ƞ 2 = .072.
We checked the extent to which more time spent cycling was associated with stronger improvement in cognitive performance. There was no cycling dose effect on any of the measures that showed improvement from cycling, so more cycling overall did not relate to greater improvement in cognitive function or well-being (see S3 File).
Effect of N-Acetyl Cysteine on Intracerebroventricular Colchicine Induced Cognitive Deficits, Beta Amyloid Pathology, and Glial Cells
Among the many factors responsible for the cognitive decline in Alzheimer’s disease, beta amyloid protein and plaque formation is crucial. This amyloid pathology is associated with activation of glial cells and oxidative stress but whether oxidative stress activates beta amyloid protein in the neurons is not clear. Further the expression of microglia is also known to vary during pathogenesis of beta amyloid plaques. The aim of the present study is to evaluate the antioxidant effect of NAC on amyloid pathology and cognition and also to investigate the link between amyloid pathology and glial cells activation. Intracerebroventricular colchicine in rats known mimics human AD in many aspects including memory loss, oxidative stress, and hyper phosphorylation of tau protein. The animal groups consisted of age matched control, sham operated, AD, and NAC treated in AD models of rats. Cognitive function was evaluated in active avoidance test beta amyloid protein, beta amyloid plaques, astrocytes, and microglia cells were quantified using immunohistochemistry in hippocampal and prefrontal cortices. Colchicine has resulted in significant cognitive loss, increased intraneuronal beta amyloid protein expression, increased reactive astrocytes, and activated microglia in all the regions of the hippocampus and prefrontal cortices. The antioxidant NAC has reversed the cognitive deficits and inhibited microglia activation but failed to inhibit BAP expression and astrocytosis. Intraneuronal BAP accumulation is deleterious and known to adversely affect cognition, but in this study in spite of intraneuronal BAP accumulation, the cognition is restored. It can be postulated that NAC might have reversed the effect of intraneuronal beta amyloid protein by acting on some downstream compensatory mechanisms which needs to be explored.
Alzheimer’s disease (AD) is a nonreversible, progressive, devastating neurodegenerative disease characterized by memory loss which is associated with neuronal loss. Its pathological features are abnormal buildup of extracellular amyloid plaques , intracellular neurofibrillary tangles , cholinergic deficiency , loss of synaptic connections , and its subsequent consequence the inhibition of neuronal signaling and neuronal loss. The inhibition of neuronal signaling in the hippocampal network is the major cause for memory loss and cognitive impairment in AD. Beta amyloid protein (BAP) is a derivative from a glycoprotein named amyloid precursor protein. Damage to neurons causes accumulation of BAP, which is due to consecutive cleavage of the APP in the cell membrane. Then by series of reaction (β-secretase and γ-secretase cleaves APP) BAP is released to the extracellular space . The disparity between BAP creation and BAP clearance is the root cause for the creation of amyloid plaques. It was believed that BAP is synthesized only in neurons however, recent finding indicates that astrocytes play a further role in AD by synthesizing significant amounts of BAP [6, 7]. Since astrocytes are numerous in the brain, even minor quantity of amyloid secretion from astrocytes could be substantial. AD is often characterized by increase in reactive astrocytes close to the sites of amyloid plaques .
Oxidative stress occurs during progression of AD in presence of BAP. Elevated levels of BAP are consisted of increased levels of oxidation products in hippocampus and cortex  of AD patients. In addition to this, the cellular stressor can increase APP expression and therefore increase BAP secretion . It is believed that the oxidative stress on the action of γ-secretase (enzyme which cleaves APP) in astrocytes is the primary cause for production of BAP  apart from other conditions. Hence boosting the antioxidant defense in astrocytes and neurons would minimize the BAP, β-amyloid plaques, and memory loss.
Whether BAP accumulation is a cause or consequence of AD remains a question. Studies targeting BAP with several γ-secretase inhibitors, which have efficiently reduced BAP levels, have been unsuccessful in clinical trials. Further, γ-secretase inhibitors are known to have adverse effects on cognition . This low efficacy of present therapeutics for the treatment of AD has guided the researchers to look at alternate avenues. BAP accumulation is mainly initiated and enhanced by oxidative stress . Several antioxidants have been testified to inhibit the formation of β-amyloid plaques or BAP and also destabilize them. In studies in transgenic mice model, some antioxidant compounds reduced plaque load in vivo . Glutathione (GSH) and thioredoxin are two intracellular antioxidants in addition to other antioxidants obtained from diet help in normalizing the ageing induced alteration [15, 16]. It has been revealed that the level of GSH is reduced in hippocampus and cortical areas of patients with AD as compared with controls . N-acetyl cysteine (NAC) is a derivative of amino acid, cysteine, and a precursor in the formation of the antioxidant glutathione in the body. NAC’s neuroprotective action is through restoration of glutathione pool  and direct scavenging ability against reactive species . However little attention has been focused on the effect of NAC on BAP pathology except for a preclinical study that provided some evidence that administration of NAC is beneficial in transgenic mouse model of AD by decreasing BAP .
Microglial cells generation is triggered in presence of β-amyloid plaques in neocortex . There is another study suggesting that beta amyloid plaques cause loss of microglial cells and inhibition of neural stem cells . BAP cause oxidative stress through microglial activation . Oxidative stress and neuroinflammation together create a malicious cycle in AD pathology . Microglial activation and oxidative stress can be increased as a result of BAP formation. Therefore, mediations that reduce BAP-induced microglial activation and oxidative stress might be useful for AD treatment. If an antioxidant treatment prevents plaque formation, thereby reducing the microglial expression or enhancing the reactive microglial expression for phagocytosis of these plaques is not clear. Hence the present study would provide facts about the expression of microglial cells in an animal model of AD.
Although no single animal model recapitulates all of the features of the AD, each model allows for in-depth analysis of only one or two components of the disease. Colchicine, a microtubule distracting agent, causes damage of neurons through neurofibrillary disintegration , BAP expression , oxidative stress , and neuronal loss all these features closely simulates human AD . Moreover, the present study is aimed at looking at the effect of glutathione supplement on amyloid pathology. Hence in this experiment of an in vivo model of AD, we test the ability of NAC in minimizing BAP, β-amyloid plaques, cognitive loss, and also its effect on expression of astrocytes and microglia.
In-house bred male albino Wistar rats, four months old and weighing 250-270g, were used in this study. Rats were fed with water and food ad libitum. The rats were maintained under controlled conditions of light-dark cycle (12:12), temperature (22±3°C), humidity (50±10%), and pathogen-free environment. Polypropylene cage with paddy husk as bedding material was used for housing the rats. The experimental procedure was approved by Institution Animal Ethics Committee (IAEC/KMC/2012).
2.2. Animal Groups
The rats were randomly divided into the following five groups (n=12 in each group). i) Control- rats in this group remained in the home cage without any surgical procedure and were treated with saline throughout the experimental period (2 weeks). ii) Sham-rats in this group underwent a sham surgical procedure, where skull surface was exposed, a bur hole was drilled aiming to the lateral ventricle, a 32G needle was lowered into lateral ventricle, 5μl of sterile artificial CSF was injected and needle was withdrawn, and finally skin was sutured. These rats were treated with saline throughout the experimental period. iii) Alzheimer’s disease- (AD-) rats in this group were injected with colchicine into ventricle stereotaxically (15μg) to induce Alzheimer’s disease. These rats were treated with saline throughout the experiment. Iv) Alzheimer’s treated with 50mg/kg of NAC- (AD+NAC-50-) rats in this group were injected with colchicine into ventricle stereotaxically to induce Alzheimer’s like disease and were treated with NAC (50mg/kg, i.p.) throughout the experiment. V) Alzheimer’s treated with 100mg/kg of NAC- (AD+NAC-100-) rats in this group were injected with colchicine into ventricle stereotaxically to induce Alzheimer’s-like disease, and were treated with NAC (100mg/kg, i.p.) throughout the experiment. Rats in all groups were subjected to active avoidance learning and memory test after treatment period (2 weeks)
NAC was purchased from Lobo Chemicals (Mumbai, India). Artificial cerebrospinal fluid (ACSF: in m mol/l:147NaCl, 2.9 KCl, 1.6 MgCl2, 1.7 CaCl2 and 2.2 dextrose) was obtained from Biotech India Pvt. Ltd. (New Delhi, India). Colchicine was obtained from Sigma Aldrich (Sigma chemicals, St. Louis, MO, USA). Rabbit polyclonal anti-beta amyloid antibody (Cat#-ab2539) known to express neuronal cytoplasm as beta amyloid protein (BAP) and extra neuronal beta amyloid plaques was obtained from abcam (Cambridge, MA, USA), rabbit polyclonal antiglial fibrillary acidic protein (GFAP) for astrocytes was from Dako Flex (Cat#-IS524, Agilent Technologies India Pvt. Ltd., Bangalore, India), and rabbit monoclonal anti-Iba1 for microglia was from abcam (Cat#-ab178847, Cambridge, MA, USA). All other chemicals and reagents are HPLC or analytical grade were from Sigma Aldrich (Sigma chemicals, St. Louis, MO, USA).
2.4. Surgery and Intracerebroventricular (ICV) Administration of Colchicine
To create an Alzheimer’s-like model, colchicine (a microtubule disrupting agent, also known to cause oxidative stress) was injected into the lateral ventricle (either left or right) stereotaxically. Stereotaxic surgical procedure was as described in our previous study . Briefly, the rats were anesthetized with sodium pentobarbital (40mg/kg, i.p.) and skull was exposed with a midline skin incision. A bur hole was drilled on the skull cap at the following stereotaxic coordinate: Anteroposterior, 0.8mm behind the bregma, and lateral, 2mm from midline [30, 31]. The skull cap was drilled carefully up to the level of dura mater, without damaging any nervous tissue. A 32G needle connected to one end of a capillary tube was held in the needle holder of the stereotaxic apparatus and inserted through the bur hole to a depth of 3.2mm from skull surface aiming at the lateral ventricle. Other end of the capillary tube was connected to a Hamilton microsyringe filled with colchicine (or artificial cerebrospinal fluid for sham group). Hamilton microsyringe was positioned in an infusion pump (Harvard apparatus). 5μl of artificial cerebrospinal fluid or 15μg colchicine in 5μl of artificial cerebrospinal fluid was injected slowly over a period of 20 minutes. Needle was held in place for an additional 5 minutes before withdrawal, in order to prevent the backflow of the injected materials. Thereafter the needle was gently removed, and the scalp was closed with sutures. Antibiotics were applied on the surgical wound to prevent any infection. The rats were kept in a warm place until they recover from anesthesia. Special care was taken during the postoperative period to provide food and water inside the cage of the rat. Following surgery, the rats were housed individually in cages until end of the experiment.
2.4.1. NAC Administration
NAC in physiological saline was administered intraperitoneally, one week prior to surgery and one week following the surgery at 50mg/kg or 100mg/kg dose. The doses of NAC were selected based on earlier studies , and human dose calculated for rats. Twelve rats were used for cognitive test in each group. Out of the twelve rats six were randomly selected for immunohistochemical studies.
2.5. Active Avoidance Test
This test was employed to evaluate associative learning and memory retention, at the end of treatment period. In this test, ability of the rat to evade an aversive experience by learning to accomplish a specific behavior in response to a stimulus signal is assessed. The shuttle box apparatus used for this test was a closed wooden box with shutter doors in the front wall. The floor area consisted of a stainless-steel metal grid, separated into two compartments by a median wall with an open door interconnecting two compartments. The floor grid of the two compartments was connected to an electric stimulator. A buzzer was installed inside shuttle box to give a discriminative sound stimulus during behavioral test. Behavioral test consisted of i) exploration, ii) active avoidance learning, and iii) memory retention test. Exploration was on
day of test, where rats were placed in the box and allowed to explore both compartments of the apparatus for 5min to make them familiar with shuttle box. Active avoidance learning test was for five consecutive days. Rat was placed in one of the compartments and allowed to explore both compartments for five minutes. At the end of exploration, a discriminative sound stimulus was provided through the buzzer, during which the rat could move to other compartment in order to avoid the foot shock. If the rat failed to move to other compartment, as soon as it hears the discriminative sound stimulus, a foot shock (2.5mA) was delivered through the grid floor for a maximum of 10 seconds, during which it could cross to the other compartment and escape the foot shock. The possibility for avoiding the foot shock was a single crossing over to other compartment of the shuttle box. The active avoidance learning consisted of 30 trials/day for 5 consecutive days. The ‘number of shock avoidances/day’ (total number of shock avoidances in 5 days divided by 5) was calculated for each rat. In the initial trials, rats fail to associate the sound stimulus to shock avoidance, but in the subsequent trails they learned to associate sound stimulus to shock avoidance by actively moving to the other compartment. In the control rats, the number of shock avoidances increases from day 1 to 5 of the test. Any decrease in the number of shock avoidances/day is an indication of learning impairment. Memory retention test was done one week after the last learning trial to assess memory retention. Memory retention test was similar to the learning trails but for only one day. A comparison of rat’s performance in learning trails with its performance during memory retention gives the assessment of memory and is presented as the percentage of memory retention score (% RTS), which was calculated by using the following formula . Any decrease in the % RTS and number of shock avoidances/day during retention test is an indication of memory impairment.
2.6. Tissue Processing for Immunostaining
A day after the active avoidance test, six rats from each group were deeply anesthetized with ether and perfused with saline followed by freshly prepared 4% paraformaldehyde in phosphate buffer (pH=7.4). The brains were postfixed in 4% paraformaldehyde in phosphate buffer for 48 hours. Brain tissues were processed for paraffin blocks. Coronal sections (7μ) from prefrontal cortex and hippocampus region were cut using a rotary microtome (Jung Biocutt 2035, Lieca, Wetztar, Germany, . Five sections from each rat brain were mounted on gelatin coated slides and air dried.
2.7. Immunostaining for BAP, β-amyloid Plaques, Astrocytes, and Microglia
The paraffin sections were immunostained with anti-beta amyloid antibody for expression BAP in the neurons and beta amyloid plaques outside the neurons , antiglial fibrillary acidic protein (GFAP) for astrocytes , and anti-Iba1 for microglia in the prefrontal cortical regions and hippocampal subregions. Sections were deparaffinized in xylene and rehydrated in descending grades of ethyl alcohol. Antigen retrieval was done by incubating the sections in 0.1M citrate buffer at 60°C for 30 min. The sections were treated with 3% H2O2 for 30 minutes to reduce the endogenous peroxidase activity in the tissue. Sections were then incubated for 30 minutes with 5% normal goat serum along with 0.3% Triton X-100 in PBS (pH7.4) to block the nonspecific binding of the primary antibody. The sections were incubated with rabbit polyclonal anti-beta amyloid antibody (1:500) or rabbit polyclonal antiglial fibrillary acidic protein (GFAP, 1:500) or rabbit monoclonal anti-Iba1 (1:1000) overnight at 4°C. The sections were incubated with biotinylated goat-anti-rabbit IgG as secondary antibody (1: 200, Vector Laboratories, Burlingame, CA) for 1hr at room temperature. The sections were washed in PBS and treated with avidin-biotin-peroxidase complex (ABC kit, Vector Laboratories, Burlingame, CA) for 1hr at room temperature. Subsequently color was developed with 3,3′-diaminobenzidine as chromogen (DAB, Vector Laboratories, Burlingame, CA). Throughout the staining protocol, sections were washed three times with PBS after each incubation. To assess nonspecific staining, several sections in each experiment were incubated in buffer without primary antibody. Sections were lightly counterstained with hematoxylin, dehydrated in ascending ethanol grades, cleared in xylene, and cover slipped in Permount (Fisher Scientific, Pittsburgh, PA).
2.8. Quantification of BAP Positive Neurons, β-amyloid Plaques, Astrocytes, and Microglia
High quality images were captured with 40x objective, with an Olympus digital camera (DP75) attached to an Olympus microscope. In each image immunostained BAP positive neurons or GFAP positive astrocytes or Iba1 positive microglia were counted using NIS Elements Br version 4.30 software. In each hippocampal section, 300 μm length of Cornu Ammonis subregions (CA1, CA2, CA3, and CA4) and 300 μ 2 area of the dentate gyrus (DG) were selected for quantification. In prefrontal cortex, the number of neurons/astrocytes/microglia in 300 μ 2 area was counted in medial, lateral, and orbital regions of the prefrontal cortex. Slides from different groups of rats were coded to avoid manual bias while counting the cells.
2.9. Statistical Analysis
The data were expressed as mean ± SE and were analyzed with one-way ANOVA, followed by Bonferroni’s multiple comparison post hoc test useing SPSS (version 25) statistical analysis software. P values <0.05 were considered as significant. The treatment effect between 2 doses of NAC was assessed through paired Student’s t-test.
3.1. Memory Retention Test
The mean number of shock avoidance/day (mean of 5 days’ avoidance), mean number of shock avoidance during retention test and % retention score (%RTS) did not differ between control and sham operated rat groups. ICV colchicine (AD like disease in rats) had significantly reduced mean number of avoidance/day during learning (p<0.01), during memory retention test (p<0.001) and % retention score (p<0.001), clearly demonstrating learning disability and memory impairment (Figures 1(a)–1(c)). NAC treatment (at both the doses) in AD model of rats has significantly increased mean number of avoidance/day during learning (Figure 1(a), p<0.001), during memory retention test (p<0.001, Figure 1(b)) and % retention score (p<0.05, Figure 1(c)) compared to rats receiving only colchicine. The number of shock avoidances/day during learning and during memory retention test and % retention score (%RTS) did not differ between control rats and AD + NAC group of rats. There was no significant (p>0.05) dose-dependent effect on any of the parameters studied. This is an indication that IVC colchicine induced cognitive loss and poor memory retention was almost reversed and brought back to the control levels (Figures 1(a)–1(c)).
Performance of rats in different groups in active avoidance learning (a) and in memory retention test ((b) and (c)). Note that AD rats had significant learning and memory deficit and deficits were decreased/normalized by treatment with NAC (both 50mg/kg and 100mg/kg). Control/sham versus AD: ∗∗∗, p<0.001 AD versus AD+NAC50 or AD versus AD+NAC100: ###, p<0.001 (One-way ANOVA, Bonferroni’s multiple comparison test, n=6 in all groups).
3.2. BAP Positive Neurons and β-amyloid Plaque Expression
Amyloid plaques were sparsely distributed in the AD model of rats. They were of diffuse variety and very rarely compact or dense-cored plaques were seen. Positive intracellular staining for BAP was observed in AD model of rats. Amyloid deposits in blood vessels were abundant in many areas of the brain investigated including hippocampal subregions (Figure 2). Cannula implantation has not significantly (p>0.05) affected the total number of BAP positive neurons compared to control rats in any regions of the hippocampus or prefrontal cortices. The number of BAP positive cells were significantly (p<0.001) high in all the regions of the hippocampus in AD model of rats compared to control or sham operated group of rats (Figure 3). Neurons in the prefrontal cortex also expressed β-amyloids extensively and the number of BAP positive cells was significantly high in AD model of rats compared to control or sham operated group of rats (p<0.001, Figure 4). NAC treatment at 50 or 100mg/kg dose showed a significant increase in BAP positive cells compared to control or sham operated groups (p<0.001, Figures 3 and 4). Further there was no significant difference in number of BAP positive cells expression between AD group of rats compared to AD rats who received NAC in all the regions of the hippocampus except in CA1 region. Similar results were observed in prefrontal cortices also (Figure 4). In CA1 region, the BAP positive cells were significantly more in AD rats treated with NAC compared to AD rats.
Photomicrographs of the hippocampal subregions in different groups of rats immunostained for β-amyloid protein (BAP). Note the expression of BAP (arrow) in large number of neurons in AD group in all regions. The number of BAP positive neurons was less or not present in NAC treated groups (AD+NAC 100) (photomicrographs of sham and AD+NAC 50 groups were avoided for simplicity). Scale bar= 25μ, in CA1-CA4 regions, =15μ in dentate gyrus (DG).
Quantitative estimation of number of neurons expressing BAP in various regions of the hippocampus. In CA1, CA2, CA3, and CA4 regions 350 μm length and in dentate gyrus (DG) 150μ 2 area were selected for quantification. Note that, in all regions, number of neurons expressing BAP significantly increased in AD and AD+NAC 50, AD+NAC 100 groups compared to control group. Values are expressed as mean ± SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001 (one-way ANOVA, Bonferroni’s multiple comparison test, and n=6 in all groups).
Photomicrographs of the prefrontal cortical regions in different groups of rats immunostained for β-amyloid protein (BAP). Note the expression of BAP (Arrow) in large number of neurons in AD group in all regions. The number of BAP positive neurons was less or not present in NAC treated groups (AD+NAC 100) (photomicrographs of sham and AD+NAC 50 groups are avoided for simplicity). Scale bar= 25μ. Graph shows quantitative estimation of number of neurons expressing BAP in frontal cortical regions. In all regions 300μ 2 area was selected for quantification. Note that, in all regions, number of neurons expressing BAP significantly increased in AD and AD+ NAC 50, AD+NAC 100 groups compared to control group. Values are expressed as mean ± SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001 (one-way ANOVA, Bonferroni’s multiple comparison test, and n=6 in all groups). MFC-Media prefrontal cortex, OFC-Orbital prefrontal cortex, and LFC-Lateral prefrontal cortex.
3.3. GFAP Expression
Distribution of GFAP positive astrocytes in the hippocampal subregions and in the dentate gyrus is shown in Figure 5. The density of reactive astrocytes was higher in AD and AD+ NAC group of rats in all regions compared to control rats. The reactive astrocytes were expressed abundantly at the area surrounding the neurons of hippocampus and also adjacent areas. In AD model of rats and also AD model who received NAC, the astrocytes were expressed with sharp dendritic margins which are not observed in control rats. Cannula implantation has not significantly (p>0.05) affected the astrocytes expression compared to control rats in any regions of the hippocampus or prefrontal cortices. The number of GFAP positive cells were significantly high in all the hippocampal subregions in AD models of rats compared to their control counterpart (p<0.001, Figure 6). Further NAC treatment (both the doses) in AD rats did not show any significant difference in GFAP expression compared to AD models of rats in the hippocampal subregion.
Photomicrographs of the hippocampal subregions in different groups of rats immunostained for glial acidic protein (GFAP). Note the expression of GFAP (arrow) in large number in AD and AD+NAC group in all regions. The number of GFAP positive astrocytes was more in AD and AD+NAC treated groups (AD+NAC 100) (photomicrographs of sham and AD+NAC 50 groups are avoided for simplicity). Scale bar= 25μ, in CA1-CA4 regions, =15μ in dentate gyrus (DG).
Quantitative estimation of number of GFAP positive astrocytes in various regions of the hippocampus. In CA1, CA2, CA3, and CA4 regions 350 μm length and in dentate gyrus (DG) 150μ2 area were selected for quantification. Note that, in all regions, the number of astrocytes was significantly increased in AD and AD+ NAC 50, AD+NAC 100 groups compared to control group. In dentate gyrus, number of astrocytes in AD+NAC-100 significantly decreased compared to AD group. Values are expressed as mean ±SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001, AD versus AD+NAC 100: #, p< 0.05 (one-way ANOVA, Bonferroni’s multiple comparison test, and n=6 in all groups).
The pattern of distribution of GFAP positive astrocytes in the prefrontal cortical regions is shown in Figure 7. As in the hippocampus and dentate gyrus, the astrocytes are densely packed in AD and AD+ NAC groups compared to control rats. GFAP positive cells in AD and AD+NAC were significantly (p<0.001) high in all the three prefrontal cortices examined compared to their control counterparts (Figure 7). However, sham operated group did not show any significant increase in GFAP expression compared to control. NAC at both doses did not alter GFAP expression compared to AD model of rats. It was also observed that the GFAP expression in NAC treated AD models of rats was significantly high compared to control group of rats (p<0.001, Figure 7).
Photomicrographs of the prefrontal cortical regions in different groups of rats immunostained for glial acidic protein (GFAP) to label the astrocytes. Note large number of astrocytes in AD and AD+NAC group in all regions. The number of GFAP positive astrocytes was more in AD and AD+NAC treated groups (AD+NAC 100) (photomicrographs of sham and AD+NAC 50 groups are avoided for simplicity). Scale bar= 25μ). Graph shows quantitative estimation of number of GFAP positive astrocytes in various regions of the prefrontal cortex. In all regions 300μ2 area was selected for quantification. Note that, in all regions, the number of astrocytes was significantly increased in AD and AD+ NAC 50, AD+NAC 100 groups compared to control group. Values are expressed as mean ±SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001 (one-way ANOVA, Bonferroni’s multiple comparison test, and n=6 in all groups). MFC-Media prefrontal cortex, OFC-Orbital prefrontal cortex, and LFC-Lateral prefrontal cortex.
3.4. Expression of Iba1 Positive Microglia
The Iba1 positive microglia were found abundantly at the area away from the neurons of hippocampus and prefrontal cortex unlike astrocytes expression which are more closely associated with neurons (Figures 8 and 10). Cannula implantation has not significantly (p>0.05) affected the microglia expression compared to control rats in any regions of the hippocampus or prefrontal cortices. The number of activated microglia was significantly high in AD model of rats compared to control or sham operated group in all the regions of the hippocampus (p<0.001, Figure 9). This indicates that ICV colchicine induces activation of microglia. NAC treatment at both doses in AD rats has significantly reduced the expression of activated microglia compared to AD model of rats in all the regions of the hippocampus (p<0.001, Figure 9). This is an indication that NAC has exerted its neuroprotective effect by minimizing the gliosis. The quantitative data on activated microglia in AD model of rats treated with either 50 or 100 mg/kg dose of NAC did not show any statistically significant (p>0.05) difference with control group of rats in CA1 and CA4 regions. However, in CA2, CA3, and DG regions, the AD model of rats treated with NAC (either 50 or 100mg/kg dose) expressed a significantly (p<0.001) higher number of reactive microglia compared to control rats. This can be assumed that the NAC has exerted its restored effect in CA1 and CA4 regions compared to the remaining subregions of the hippocampus. The expression of activated microglia was significantly more in AD model of rats compared to control or sham operated rats in all the three regions of the prefrontal cortex investigated (p<0.001, Figure 10). This indicates that ICV colchicine has caused microgliosis in prefrontal cortices. NAC treatment (either 50 or 100 mg/kg dose) in AD model of rats significantly reduced the expression of activated microglia in all the three prefrontal cortices compared to AD model of rats (p<0.001, Figure 10). This indicates the neuroprotective effect of NAC against colchicine induced microgliosis. Further in the comparison between AD rats treated with NAC and control rats, the MFC regions showed a significant difference but not in the LFC and OFC (p<0.001, Figure 10). This indicates that NAC has exerted its protective effect better in LFC and OFC compared to MFC.
Photomicrographs of the hippocampal subregions in different groups of rats immunostained for Iba1 to label reactive microglia. Note the presence of reactive microglia (Arrow) in large number in AD group in all regions and their distribution is less dense in AD+NAC group (photomicrographs of sham and AD+NAC 50 groups are avoided for simplicity). Scale bar= 25μ, in CA1-CA4 regions, =15μ in dentate gyrus (DG).
Quantitative estimation of number of Iba1 positive reactive microglia in various regions of the hippocampus. In CA1, CA2, CA3, and CA4 regions 400μm length and in dentate gyrus (DG) 400μ2 area were selected for quantification. Note that, in all regions, the number of microglia was significantly increased in AD group compared to control group. Number of microglia decreased significantly in AD+NAC 50 and AD+NAC 100 compared to AD group. In CA2, CA3, and DG number of microglia significantly increased compared to control group. Values are expressed as mean ± SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001, AD versus AD+NAC 50/100: ###, p< 0.001 (one-way ANOVA, Bonferroni’s multiple comparison test, n=6 in all groups).
Photomicrographs of the frontal cortical regions in different groups of rats immunostained for Iba1 to label reactive microglia. Note the presence of reactive microglia (arrow) in large number in AD group in all regions and their distribution is less dense in AD+NAC group (photomicrographs of sham and AD+NAC 50 groups are avoided for simplicity). Scale bar= 25μ. Graph shows quantitative estimation of number of Iba1 positive reactive microglia in various prefrontal cortical regions. In all regions 400μ2 area was selected for quantification. Note that, in all regions, the number of microglia was significantly increased in AD group compared to control group. Number of microglia decreased significantly in AD+NAC-50 and AD+NAC 100 compared to AD group. In MCF, number of microglia significantly increased compared to control group. Values are expressed as mean ±SE. Control/sham versus AD: ∗∗∗, p<0.001 control/sham versus AD+NAC50 or control/sham versus AD+NAC100: $$, p<0.001, AD versus AD+NAC 50/100: ###, p< 0.001 (one-way ANOVA, Bonferroni’s multiple comparison test, and n=6 in all groups). MFC-Media prefrontal cortex, OFC-Orbital prefrontal cortex, and LFC-Lateral prefrontal cortex.
The major findings of this experiment are that a week after ICV colchicine administration to rats resulted in severe cognitive deficit. Immunohistochemistry conducted in these rats after cognitive test (about 26 days after colchicine administration) revealed a significantly higher number of BAP positive neurons in the hippocampus and prefrontal cortices, the areas concerned with cognition. Further this is accompanied with gliosis involving both astrocytes and microglia in hippocampus and prefrontal cortices. The cognitive deficits observed a week after colchicine administration involves amyloid pathology and gliosis in hippocampus and prefrontal cortices. The upregulated intraneuronal BAP expression, which is more toxic than plaques, and the neuroinflammation observed by gliosis is the factors responsible for cognitive impairment. NAC treatment a week before and a week after colchicine administration has reversed the cognitive deficits. However, NAC treatment did not alter the intraneuronal BAP expression and also the expression of reactive astrocytes in hippocampus and prefrontal cortices. However, the activated microglia expression was downregulated after NAC administration. The reversal of cognitive deficits after NAC treatment in AD model of rats is associated with reduction in microglia expression but not BAP positive neurons or expression of reactive astrocytes.
Many transgenic AD animal models which expressed accumulation of BAP inside and outside the neurons showed severe cognitive deficits . In our study, the colchicine treated AD model of rats exhibited similar intraneuronal BAP in cortical and hippocampal areas which is associated with cognitive impairment. ICV colchicine induced oxidative stress and concomitant increase in BAP level of hippocampal tissue was demonstrated before [36, 37]. ICV colchicine is also known to cause excessive free radical generation and oxidative damage . Oxidative stress induced BAP accumulation and resultant cognitive loss were reported earlier [39, 40]. Hence cognitive deficits in the form of disability in learning and memory observed in this study can be positively correlated to BAP and probably the oxidative stress, though we did not measure the oxidants in the brain. In this study, enhancing the brain antioxidant capacity by NAC treatment was able to reverse the cognitive deficits. It is well-known that oxidative stress is responsible for changes in the neurons and behavioral deficits in AD . In previous studies, NAC has reversed behavioral deficits observed in traumatic brain injury in several animal models [42–44] due its antioxidant potential. NAC treatment in mice receiving ICV injections of BAP had improved learning and memory compared to vehicle-treated animals . This study claims that the cognitive deficits observed is due to upregulation of BAP expression and this part of their findings was similar to our findings in this study. However, unlike their study, we noticed that NAC treatment has reversed the cognitive deficits in spite of higher level of BAP expression in hippocampal and prefrontal cortices. This is an interesting finding but it requires further studies to evaluate this phenomenon. We can only assume that the choice of behavioral study model used in our study evaluates a form of procedural memory which is likely to be affected only in late stages of AD. It can also be postulated that NAC might have reversed the effect of intraneuronal beta amyloid protein by acting on some downstream compensatory mechanism which needs to be explored. NAC may have a role in disintegration of BAP and beta amyloid plaques. Production of BAP within the neurons results from two proteases cleaving APP: β-secretase and γ-secretase. NAC is known to inhibit APP gene transcription . It has significantly decreased soluble levels of BAP in transgenic mice that overexpress the APP gene . There are many studies that demonstrate that NAC is capable of curbing amyloid pathology in AD model of rats. However, our study did not demonstrate any noticeable effect of NAC in amyloid pathology. Hence the beneficial effect of NAC observed in reversing the cognitive deficits involves higher complex mechanisms and least likely due to amyloid pathology alone.
The neuroglial interactions are key in neurotransmission and any interruption in glial functions also contribute to cognitive dysfunctions. Microglia and astrocytes are activated in regions of the brain occupied by amyloid plaques and also oxygen free radicals [47, 48] which are characteristics of AD. In AD, there is an increase in the number of activated microglia and reactive astrocyte close to the sites of amyloid plaques . These reactive astrocytes surrounding beta amyloid plaques are the cause for local inflammatory response and it modifies calcium signaling [49, 50]. Loss of astroglial function and reactivity contribute to neurodegenerative diseases like AD . In this study we observed activation of astrocytes and higher number of reactive microglia expression (microgliosis) in almost all the areas of hippocampus and prefrontal cortices in presence of BAP positive neurons. Transcription of proinflammatory cytokines and chemokines occur due to BAP-induced intracellular signaling pathways. This can result in cellular damage to the astrocytes or even stimulate BAP in astrocytes . Astrocytes appear to be the chief target of BAP, as this protein brings several effects of oxidative stress including defective intracellular calcium signaling . Though we could not demonstrate BAP in astrocytes or altered calcium signaling, activation of astrocytes was very much evident in all the areas we investigated. Apart from this, the gliosis observed in this study could be due to colchicine induced neuroinflammation and neuronal damage or oxidative stress.
Adequate evidence suggests that amyloid plaques are not randomly distributed in the brain but show a characteristic spatial pattern. Studies showed that CA1 is one of the most affected regions in AD, mainly at early stages. In our study ICV colchicine has resulted in BAP expression in all the subregions of the hippocampus which also showed severe gliosis. In addition to that in CA1 region the number of BAP positive cells was more in AD model of rats who received NAC compared to rats who received only colchicine.
In our previous study we demonstrated that ICV colchicine causes upregulation of hyper phosphorylated tau protein with considerable neuronal loss and cognitive decline. In the same experiment NAC administration has reversed tau pathology, cognitive deficits, and also the neuronal loss . Correlating with earlier findings, it can be postulated that NAC can reverse tau pathology but not amyloid pathology. The reversal of cognitive function may be due to downregulation of tau protein expression in hippocampus and prefrontal cortices and also reduced expression of activated microglia. It is largely believed that the tau aggregation is probably induced by b-amyloids and neurofibrillary tangles appear in the brain later than senile plaques. However, it has also been suggested that accumulation of Aβ plaques does not correlate with cognitive impairments in AD patients. A large number of individuals without any cognitive impairment accumulate Aβ plaques in their brains [55, 56]. Another interesting factor is that β-amyloid plaque accumulation is not intrinsically cytotoxic and also that BAP does not induce tau accumulation . In a recent review article by Kametani and Hasegawa  they claimed that AD is a disorder that is triggered by impairment of APP metabolism and progresses through tau pathology, not Aβ amyloid.
Astrocytes are involved in maintaining or processing oxidative stress in AD. Astrocytes have a key role in maintaining the neuronal integrity damaged or activated astrocytes are vulnerable to neuronal functions. Thus activated astrocytes observed in this study might have caused oxidative stress and inhibited axonal transmission which resulted in cognitive dysfunction. It can also be correlated that overexpression of BAP, as observed in this study, has caused oxidative stress in neurons as well as astrocytes. Excessive BAP is known to induce oxidative stress in brain . Astrocytes are the producers of the raw materials needed for the production of glutathione in neurons . It can be assumed that upregulation of BAP in astrocytes could prevent glutathione production. Preclinical data also provide evidence that NAC treatment is beneficial in AD murine models counteracting oxidative damage . Further Tucker and coworkers  demonstrated antiamyloid efficacy of NAC. However, in our study supplementation of NAC, the glutathione precursor did not alter the BAP expression. Hence one of the reasons for continued higher expression of BAP is due to continued higher expression of activated astrocytes. The bioavailability of glutathione is poor (low solubility and absorption, together with a rapid metabolism and elimination) hence testing with higher dose of NAC and measuring glutathione content from hippocampus would likely provide better understanding.
Microglial cells phagocytose beta amyloid plaques, as they express beta amyloid plaque degrading enzymes. They also get activated to produce inflammatory chemokines, cytokines, and neurotoxins . It is suggested that microglial cells play a duel role in the pathogenesis of AD . They are able to clear soluble fibrillar Aβ however their constant interactions with Aβ can cause an inflammatory response thereby resulting in neurotoxicity. In this study we observed microglial activation in hippocampus and prefrontal cortices. NAC is known to exert its neuroprotective potential through two well-known mechanisms, that is, restoration of glutathione pool  and direct scavenging ability against reactive oxygen species . Activation of microglia is a hallmark of neuroinflammation, which enhances the production and release of reactive oxygen species. NAC, the antioxidant, is involved in detoxification of reactive oxygen species in the brain. Hence it is supposed that NAC would inhibit microglial activation. Accordingly, in our study, NAC has inhibited microglial activation in hippocampus and prefrontal cortices in presence of BAP positive neurons. This suggests that oxidative stress induced by colchicine is responsible for microglial activation. BAP initiates a cascade of events, including activation of microglial cells and oxidative stress . But in our study the activation of microglia was inhibited by NAC even in presence of BAP positive neurons. In a rat model of spinal cord injury both astrocytes and microglial expressions were increased . NAC treatment in these rats had no effect on reactive astrocytes but the microglial reaction was significantly decreased. From these findings it can be suggested that NAC has a significantly positive effect on microglia but not on astrocytes. This is possible because the astrocytes itself might have expressed abundant BAP, since NAC has not been able to minimize the BAP expression. Further the response of astrocytes and microglia may be different in presence of amyloid pathology. Evaluating the expression of BAP in astrocytes would likely provide further information.
ICV colchicine causes intraneuronal BAP expressions and cognitive loss which is associated with gliosis. The antioxidant NAC has reversed the cognitive deficits and inhibited microglia activation but failed to inhibit expression of BAP positive neurons and reactive astrocytes in an animal model of AD. It can be postulated that NAC might have reversed the effect of intraneuronal beta amyloid protein by acting on some downstream compensatory mechanism which needs to be explored.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors have not declared any conflicts of interest.
Sampath Madhyastha conceived the idea and designed and supervised the experiment. Teresa Joy performed the experiments and analyzed the data. Muddanna S. Rao supervised immunohistochemistry.
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Copyright © 2019 Teresa Joy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
GlyNAC improves multiple defects in aging to boost strength and cognition in older humans
A pilot human clinical trial conducted by researchers at Baylor College of Medicine reveals that supplementation with GlyNAC -- a combination of glycine and N-acetylcysteine as precursors of the natural antioxidant glutathione -- could improve many age-associated defects in older humans to improve muscle strength and cognition, and promote healthy aging.
Published in the journal Clinical and Translational Medicine, the results of this study show that older humans taking GlyNAC for 24 weeks saw improvements in many characteristic defects of aging, including glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, body fat, genomic toxicity, muscle strength, gait speed, exercise capacity and cognitive function. The benefits declined after stopping supplementation for 12 weeks. GlyNAC supplementation was well tolerated during the study period.
"There is limited understanding as to why these defects occur in older humans, and effective interventions to reverse these defects are currently limited or lacking," said corresponding author endocrinologist Dr. Rajagopal Sekhar, associate professor of medicine in the Section of Endocrinology, Diabetes and Metabolism at Baylor.
For the last 20 years, Sekhar and his team have been studying natural aging in older humans and aged mice. Their work brings mitochondria, known as the batteries of the cell, as well as free radicals and glutathione to the table in discussions about why we age.
Mitochondrial dysfunction and aging
Mitochondria generate energy needed for supporting cellular functions by burning fat and sugar from foods, therefore mitochondrial health is critically important for life. Sekhar believes that improving the health of malfunctioning mitochondria in aging is the key.
As mitochondria generate energy, they produce waste products such as free radicals. These highly reactive molecules can damage cells, membranes, lipids, proteins and DNA. Cells depend on antioxidants, such as glutathione, the most abundant antioxidant in our cells, to neutralize these toxic free radicals. Failing to neutralize free radicals leads to harmful and damaging oxidative stress that can affect mitochondrial function.
Interestingly, glutathione levels in older people are much lower than those in younger people, and the levels of oxidative stress are much higher.
Animal studies conducted in the Sekhar lab have shown that restoring glutathione levels by providing GlyNAC reverses glutathione deficiency, reduces oxidative stress and fully restores mitochondrial function in aged mice.
"In previous work we showed that supplementing HIV patients with GlyNAC improved multiple deficits associated with premature aging observed in those patients," Sekhar said. "In this study, we wanted to understand the effects of GlyNAC supplementation on many age-associated defects in older adults."
GlyNAC improves several hallmark defects in aging
The world population of older humans is rapidly increasing and with it comes an increase in many age-related illnesses. To understand what causes unhealthy aging, scientific research has identified nine hallmark defects which are believed to contribute to the aging process.
"It is believed that correcting these aging hallmarks could improve or reverse many age-related disorders and help people age in a healthier way," Sekhar said. "However, we do not fully understand why these hallmark defects happen, and there are currently no solutions to fix even a single hallmark defect in aging."
This is where Sekhar's trial results become encouraging, because GlyNAC supplementation for 24 weeks appears to improve four of the nine aging hallmark defects.
To further understand whether GlyNAC holds the keys to mitochondrial recovery and more, Sekhar and his team conducted this pilot clinical trial.
"We worked with eight older adults 70 to 80 years of age, comparing them with gender-matched younger adults between 21 and 30 years old," Sekhar said. "We measured glutathione in red-blood cells, mitochondrial fuel-oxidation, plasma biomarkers of oxidative stress and oxidant damage, inflammation, endothelial function, glucose and insulin, gait-speed, muscle strength, exercise capacity, cognitive tests, gene-damage, glucose-production and muscle-protein breakdown rates and body composition. Before taking GlyNAC, all these measurements were abnormal in older adults when compared with those in younger people."
The older participants took GlyNAC for 24 weeks, and then stopped it for 12 weeks. Sekhar and his colleagues repeated the above measurements at the halfway point at 12 weeks, after 24 weeks of taking GlyNAC, and again after stopping GlyNAC for 12 weeks.
"We are very excited by the results," Sekhar said. "After taking GlyNAC for 24 weeks, all these defects in older adults improved and some reversed to the levels found in young adults." The researchers also determined that older adults tolerated GlyNAC well for 24 weeks. The benefits, however, declined after stopping GlyNAC supplementation for 12 weeks.
"I am particularly encouraged by the improvements in cognition and muscle strength," Sekhar said. "Alzheimer's disease and mild cognitive impairment (MCI) are serious medical conditions affecting memory in older people and leading to dementia, and there are no effective solutions for these disorders. We are exploring the possibility that GlyNAC could help with these conditions by conducting two pilot randomized clinical trials to test whether GlyNAC supplementation could improve defects linked to cognitive decline in Alzheimer's disease and in MCI, and possibly improve cognitive function."
"The overall findings of the current study are highly encouraging," Sekhar said. "They suggest that GlyNAC supplementation could be a simple and viable method to promote and improve healthy aging in older adults. We call this the 'Power of 3' because we believe that it takes the combined benefits of glycine, NAC and glutathione to reach this far reaching and widespread improvement. We also have completed a randomized clinical trial on supplementing GlyNAC vs. placebo in older adults and those results will be forthcoming soon."