Evaluating Alzheimer’s disease through a better understanding of GAD67

Written By: Abel B. Cortinas

Original Article: Wang et al. Molecular Neurodegeneration 2017
The Gist of It:
Alzheimer’s disease (AD) is a complex, irreversible, and usually age-related brain disorder that slowly destroys one’s memory and thinking skills. Although it is difficult to accurately measure the exact number of people affected, the US National Institute on Aging estimates that approximately 5.5 million Americans have Alzheimer’s dementia, with most of them age 65 and older. There are currently five prescription drugs approved by the US Food and Drug Administration to treat the cognitive symptoms (that is, memory loss, confusion, and problems with thinking and reasoning) of AD. Unfortunately, there is no cure or disease-modifying therapy to treat the actual cause of AD, in large part because of the complexity of the disease itself. And, although there are currently over 130 drugs in clinical trials for the treatment of AD, the failure rate of all drugs over the past 15 years in AD research is daunting (greater than 99%!). Researchers at both Pennsylvania State University and South China Normal University studied a specific protein, GAD67, known to be involved in signaling pathways in the brain and linked to a number of other neurological disorders (like epilepsy and schizophrenia). Using mice that developed AD but also had less of the GAD67 protein, researchers revealed three key findings. First, reducing the amount of the Gad67 gene (which is responsible for making the GAD67 protein) present led to a significant decrease in the amount of a different protein called amyloid beta. Amyloid beta is notoriously known for clumping up and forming plaques in the brains of patients with AD. Second, having less of the GAD67 protein resulted in less inflammation in the brain as well as lower amounts of an important signaling molecule in the brain called gamma amino-butyric acid, or GABA for short. Lastly, these mice partially recovered their sense of smell and memory association, a condition known as the olfactory (that is, the sense of smell) sensory deficit, which is a reported symptom in patients with AD. Together, these findings suggest that the Gad67 gene and protein play an important role in the development and progression of AD. This result provides a potential strategy for treatment through inhibition or gene silencing / editing.
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THE SELECTIVE KNOCKDOWN OF THE Gad67 GENE IN MICE POTENTIALLY PLAYS AN IMPORTANT ROLE IN MODULATING THE EFFECTS OF ALZHEIMER’S DISEASE, A DISEASE THAT CURRENTLY HAS NO APPROVED THERAPIES FOR TREATMENT

The Nitty Gritty:
Wang et al. created an Alzheimer’s disease (AD) mouse model with Gad67 haploinsufficiency, referred to as AD+GG+ mice, by crossing GAD67-GFP knock-in mice with 5xFAD mice that had five human familial AD mutations in amyloid precursor protein and presenilin-1 genes. The researchers found that Gad67 haploinsufficiency resulted in significantly lower amounts of amyloid beta (Aβ) burden in the AD+GG+ bigenic mice compared with amounts in 5xFAD mice. Aβ42 immunostaining at the frontal cortex as well as quantitative detection using an Aβ ELISA protocol in the olfactory bulb and the piriform cortex demonstrated downregulated Aβ production in Gad67 haploinsufficient 5xFAD mice. Quantitative analysis of Aβ42 in the hippocampus demonstrated a slight reduction in the amount of Aβ in AD+GG+ mice, but the reduction in the hippocampus was not as significant as in the cortical areas. Further immunostaining with thioflavin-s verified that Aβ deposits were reduced in both the frontal cortex and piriform cortex of the bigenic mice. The effect of Gad67 haploinsufficiency on both GABA production and electrophysiological tonic GABA currents in astrocytes was also studied. Immunostaining revealed significantly less astrocytic GABA in the Gad67 haploinsufficient bigenic mice compared with normal 5xFAD mice. Quantitative analyses performed in the frontal cortex, piriform cortex, and hippocampal CA1 regions demonstrated a uniform reduction in astrocytic GABA. Contrastingly, neuronal GABA was not significantly changed across all groups of mice. Subsequently, whole-cell patch-clamp recordings were performed on the layer IV-VI cortical neurons to determine whether reduced GABA content resulted in reduced tonic GABA currents. They found that Gad67 haploinsufficiency resulted in a reduction of the abnormal GABA tonic currents in cortical neurons of 5xFAD mice. Next, microglia reactivity was assessed. Due to prior research suggesting that microglia activity is regulated by GABA production from astrocytes, immunoreactivity was measured after exposure to the proinflammatory reagent nitric oxide synthase (iNOS). In Gad67 haploinsufficient mice, the iNOS immunoreactivity was significantly reduced compared with immunoreactivity in non-AD mice in the frontal cortex and piriform cortex, as assessed by immunostaining and subsequent quantitative analysis. Lastly, Wang et al. investigated whether Gad67 haploinsufficiency rescued olfactory deficits in 5xFAD mice. Odor habituation and cross dis-habituation behavior tests were performed on all groups. The results indicated that Gad67 haploinsufficiency rescued olfactory deficits in 5xFAD mice. Taken together, Gad67 gene deficiency provided reasonable evidence to support the targeting of both the gene and its resulting protein as a potentially promising strategy to combat AD.
Original research article: Wang, Y., et al. “Gad67 haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer’s disease. 12.1 (2017): 73.

A Healthy Gut Leads to a Healthy Heart

Written by: Sayan Roychowdhury

Original article: Battson et al. American Journal of Physiology Heart and Circulatory Physiology 2019
The Gist of it:
Many people have heard that the bacteria in our gut play an important role in digestion, but did you know they can also affect your weight and cardiovascular health? Although some bacteria are associated with disease, others are vital for your immune system, heart function, and weight control. And understanding this link can be extremely important – as of 2018, almost 30% of the world’s population is considered obese; within the United States, this number is up to 40%. In overweight people, cardiovascular disease (CVD) is more than twice as likely to lead to death than it is in healthy-weight people. This increased risk is caused by stiffer arteries and restricted blood flow to the heart. Finding ways to manage the risk of CVD is crucial, especially for obese patients.
Mice that are obese have vastly different microbiomes, the population of microbes living inside their gut, than mice that are a healthy weight. This knowledge begs the question, is it possible for obese mice to have a decreased risk of dying from CVD if they have a healthy gut microbiome? A group of scientists from Colorado State University think so. Their recent paper explores the connection between the gut microbiome and physical changes to the heart and nearby arteries. These researchers exchanged the gut microbes between obese mice and healthy-weight mice and studied their cardiovascular pathology at the end of an 8-week period. They discovered that when obese mice were given gut microbes from healthy-weight mice, it reduced their cardiovascular pathology. Healthy-weight mice transplanted with the obese gut microbes displayed the opposite effects and had increased pathology. These results are exciting because they show that altering the gut microbiome could be an important strategy to combat the risk of CVD in people, in addition to diet and weight loss.
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Swapping microbes from the guts of healthy-weight and obese mice leads to changes in their cardiovascular pathology.

The Nitty Gritty:
Cecal contents were collected from two sets of mice, obese leptin-deficient (Ob) and control (Con) C57BL/6J, and used as donor samples for cecal transplants. The endogenous gut microbiota in recipient mice was suppressed prior to the transplants. Four experimental groups were created: 1) control mice given control microbiota (Con + Con), 2) control mice given obese microbiota (Con + Ob), 3) obese mice given control microbiota (Ob + Con), and 4) obese mice given obese microbiota (Ob + Ob). All groups were confirmed to exhibit the transplanted microbiota by analyzing fecal matter collected from the colon. To measure arterial stiffness, an aortic pulse wave velocity was calculated using the distance between the two probes divided by the time between the ECG R wave and the tail end of the Doppler signal. The arterial stiffness was significantly higher in mice from the Con + Ob group compared with Con + Con mice, while the stiffness in mice from the Ob + Con group was slightly lower than in mice from the Ob + Ob group. To determine the size of myocardial infarcts, no-flow global ischemia was first induced in the excised hearts, and then the hearts were reperfused for 2 hours. Finally, the left ventricle (LV) was isolated, weighed, and stained to distinguish living from infarcted tissue. LV mass was shown to be increased in both of the groups transplanted with the Ob microbiota. Infarct size was up to 20% larger in hearts from the Con + Ob group compared with hearts from the Con + Con groups, while the size in Ob + Ob hearts was significantly larger than in Ob + Con hearts. In summary, this work provides evidence that a change in gut microbiota composition can lead to modified cardiovascular pathology, independent of weight and diet.
Original research article: Battson, M.L., et al. “Gut microbiota regulates cardiac ischemic tolerance and aortic stiffness in obesity.” Am J Physiol Heart Circ Physiol 317.6 (2019): H1210–H1220.

 

 

Treating Heart Failure by “Vagus Nerve Stimulation (VNS)” implanted devices

Written By: Sravya Kotaru

Original article: Ouyang et.al. Gene 2019
The Gist of it:
The human heart pumps blood in two steps – first, blood fills into the two lower chambers of the heart called ventricles; then, the heart muscle contracts and pushes the blood out of the ventricles to other organs through blood vessels. Chronic systolic heart failure happens when the heart is not able to push enough blood out when it contracts. The main cause is an imbalance between signals that activate the heart pump and those that relax it. This results in an over-active and tired heart. Medications commonly prescribed for this type of heart failure aim to reduce the signals that activate the heart and bring back the balance. A new treatment focuses on the other side of the balance by electrically stimulating a nerve that typically relaxes the heart, called the vagus nerve. This treatment uses a device implanted under the skin near the collar bone that sends electrical signals to the vagus nerve in the neck, where it connects the brain to the heart. Scientists found that when the vagus nerve is stimulated, the heart makes increased amounts of a special type of RNA called microRNA-183-3p. MicroRNAs, unlike the more well-known mRNAs, do not contain the information to make proteins. Instead, they block certain mRNAs from making their specific proteins. MicroRNA-183-3p reduces the production of a protein called BNIP3L in the heart. In an over-active heart, BNIP3L increases the removal of mitochondria, the power plants of the heart that generate energy for its function; this removal makes the heart tire out. VNS treatment reduces the amount of BNIP3L in the heart and reverses its over-activating effects, thus protecting the heart from failure. This research has not only explained how VNS treatment is effective at the molecular level but has also opened up the possibility of developing new drugs that can mimic the effects of VNS to treat heart failure.
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Vagus nerve stimulation treats chronic systolic heart failure and increases blood output from the heart by increasing the amount of microRNA-183-3p in the heart, which in turn reduces the amount of BNIP3L protein. This restores energy generation in the heart by limiting the BNIP3L-mediated removal of the energy powerhouses, called mitochondria, thus protecting the heart from tiring out and failing.

The Nitty Gritty:
Chronic systolic heart failure (CSHF) is a complex disorder characterized by insufficient blood circulation to the lungs and other organs. A recent therapeutic method that has proven to be effective in patients with CSHF is vagus nerve stimulation (VNS), which involves electrically stimulating the parasympathetic cranial nerve X (or vagus nerve) that connects the brain to the heart, throat, and abdomen. Using an animal model that reproduced CSHF mitigation by VNS treatment in rats, Ouyang et al. investigated the molecular mechanisms of VNS action. The authors observed that VNS treatment increased the left ventricle ejection fraction (LVEF, i.e. output from systolic contraction) and decreased the left ventricular end-systolic and end-diastolic volumes (LVESF and LVEDF). VNS treatment also decreased the amount of B-type natriuretic peptide (BNP) hormone, indicating lower blood volume and heart wall extension at the end of each contraction. They observed that VNS-treated rats had higher amounts of the microRNA (miR)-183-3p and the anti-autophagy marker Bcl-2 and lower amounts of the pro-autophagy markers LC3 II/I and Beclin-1. They further observed that miR-183-3p directly silences the expression of the Bcl-2–interacting protein 3-like (BNIP3L) protein, which is known to initiate autophagy by recruiting the LC3 phagosome components. Moreover, the VNS-mediated increase in the amount of miR-183-3p led to a decrease in the amount of phosphorylation and thus inactivation of the mTOR and Akt signaling molecules. In summary, the authors discovered that VNS alleviates CSHF via the upregulation of miR-183-3p in cardiac cells and through the subsequent suppression of BNIP3L-initiated autophagy in an mTOR/Akt pathway-dependent manner.
Original Research Article: Ouyang, S., et.al. “MicroRNA-183-3p up-regulated by vagus nerve stimulation mitigates chronic systolic heart failure via the reduction of BNIP3L-mediated autophagy.” Gene (2019). doi: 10.1016/j.gene.2019.144136 [Epub ahead of print].