Adocbl Binds Directly To Lrrk2
We next tested whether AdoCbl binds directly to human LRRK2. We first used agarose functionalized with AdoCbl to pull-down purified LRRK2, which could be eluted as a function of AdoCbl . In thermal shift assays , the melting temperature of LRRK2 increased substantially from 50 to 54°C in the presence of AdoCbl . This thermostabilization by AdoCbl was comparable to that measured in the presence of the established LRRK2 kinase inhibitor PF-06447475. Furthermore, we applied microscale thermophoresis to measure the binding affinity of AdoCbl for LRRK2 and determined an apparent dissociation equilibrium constant of 12.0M and 4.1M for purified LRRK2-WT and LRRK2-G2019S, respectively , in agreement with our IC50 values. As a validation of the MST assay, under the same conditions LRRK2-G2019S binds PF-06447475 with a KD of 70nM , in line with the reported IC50 of 11nM for this inhibitor.
In addition, we examined the effects of AdoCbl using striatal slices from another preclinical LRRK2-R1441G BAC transgenic mouse model. The declined DA release evoked by 2-min intervals during a 20-min period was robust in slices from LRRK2-R1441G mice compared to WT controls, and again, AdoCbl alleviated this deficit, restoring the sustainability . Taken together, our data demonstrates that AdoCbl is capable of rescuing the impairment of DAergic neurons in evoked DA release caused by multiple LRRK2 PD mutants.
Generation Of Mouse Embryonic Fibroblasts
LRRK2-G2019S and WT control MEFs were isolated from mouse embryos at day E13.5 resulting from crosses between heterozygous LRRK2-G2019S and wild-type C57/BL/6J mice. All the MEFs were cultured for at least 30 passages to immortalize the cells. All cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine and 100units/ml Penicillin-Streptomycin, and were maintained at 37°C with 5% CO2. All cells lines were confirmed by PCR genotyping and western blot, but were not tested for mycoplasma contamination. To test inhibition, cells were treated with inhibitor or DMSO for 24h in DMEM with 3% FBS before being lysed and subject to western blot for analysis.
Compound Screen And Tr
Compounds from the FDA-Approved library were tested against flag-tagged LRRK2 purified from BAC transgenic mouse brain. LRRK2 kinase activity was monitored by measuring time resolved fluorescence resonance energy transfer emission ratio upon the phosphorylation of Fluorescein-LRRKtide and subsequent binding of Terbidium-pLRRKtide antibody . This TR-FRET-based assay was used in a high-throughput screen of small molecule chemical compounds for LRRK2 using the TECAN Freedom EVO 200 liquid handling system, and relative fluorescence was measured using the TECAN Safire 2 fluorescence spectrometer. To determine the TR-FRET ratio between Fluorescein-LRRKtide and Terbidium-pLRRKtide antibody, fluorescent intensity was measured at wavelengths 515nm and 485nm.
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C Elegans Basal Slowing Assay
Well-fed worms with intact dopaminergic neural circuitry move slower in the presence of bacterial food than in its absence. This basal slowing response was assayed as described previously., Briefly, a set of NGM assay plates were seeded with bacterial food, E. coli OP50, in a ring shape, and another set of NGM assay plates were uncoated. Age-synchronized worms were washed twice in S basal buffer . Worms were then transferred to the center of the NGM plates coated with or without E. coli OP50 as described above, settled for 5min, and their locomotion were recorded with a digital camera in 20s intervals. Body bends were examined using an unbiased machine-vision analysis system . Basal slowing was calculated as the percent slowing in body bends per 20s in the presence vs. the absence of bacterial lawn.
Treatment Of Drosophila With Adocbl
Flies were used and raised as described recently. Briefly, the TH GAL4 was crossed with either UAS-hLRRK2-wildtype, UAS-hLRRK2-G2019S or the kinase-dead UAS-hLRRK2-G2019S-K1906M line to produce progeny dopaminergic expression of the transgene . The crosses were allowed to lay eggs onto instant fly food or onto instant food supplemented with AdoCbl. The final concentration of AdoCbl in the fly food ranged from 100 to 2500nM. Females were collected on the day of emergence and transferred to new vials for 24h. In control experiments, low expression dLRRK flies were fed instant fly food or food supplemented with AdoCbl.
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The Importance Of Integrative Medicine
Integrative medicine is a particular healthcare approach that treats disease by addressing all imbalances in the body in a holistic manner. Nutrition is a primary component of integrative medicine, as the diet is largely responsible for ensuring our bodies are functioning properly. When considering the fact that nutritional deficiencies impair the way biochemical processes are carried out in the body, its crucial to address any imbalances that may contribute to the onset or outcome of chronic disease.
In recent years, researchers began taking an interest in the relationship between micronutrients and PD. For example, a newly published study assessed whether vitamin B12 deficiency is associated with impaired mobility and cognition among newly diagnosed PD patients.
Vitamin B12 And Folate
Vitamin B12 is an antioxidant. It helps keep red blood cells and nerve cells healthy and helps produce DNA. Sources of vitamin B12 are typically red meat, chicken, sardines, eggs, fortified cereals and bread, and nutritional yeast.
Researchers discovered that patients with early-onset Parkinson’s disease had lower vitamin B12 levels, which reduced motor and cognitive functions. In some cases, taking a multivitamin that included vitamin B12 slowed the loss of those functions.
Folate is found in organ meats , yeast, and leafy green vegetables. Folate plays several roles in the body and brain.
Both B12 and folate are involved in the metabolism of homocysteine, an amino acid. High levels of homocysteine are seen in various cognitive disorders. Studies show that Parkinson’s disease patients taking levodopa for the condition are also more likely to have elevated homocysteine.
In one meta-data analysis, researchers investigated the correlations between cognitive function , homocysteine, folate, and vitamin B12 levels in patients with Parkinson’s disease. They discovered that patients with cognitive dysfunction had high levels of homocysteine and lower levels of folate and vitamin B12.
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Quantitative Thermal Sensory/perception Of Temperature And Nociception
Temperature and nociception of the feet were performed by using the Thermal Sensory Analyzer TSA-2001 . The dorsum of the dominant foot was chosen as the test position. A thermode that can heat or cool the skin, as needed, was placed on the subject’s skin at the starting temperature of 32°C. During the process, the subject can perceive changes of stimulations with the temperature increased or decreased continuously at a rate of 1°C per second . Four sensory sub-modalities were tested: warm sensation , cold sensation , heat-induced pain , and cold-induced pain . Each subject completed six trials in a random order. In the process of induced pain assessments, subjects were required to respond by pressing the stop button once the thermode reached a temperature that was too hot or too cold for them to comfortably tolerate.
Findings Could Help Inform Development Of New Lrrk2
- New York, NY
The basic micronutrient vitamin B12 inhibits the activity of a protein implicated in LRRK2-associated Parkinsons disease the most common inherited form of the condition, according to research conducted at the Icahn School of Medicine at Mount Sinai and published March 11 in the journal Cell Research. The study findings suggest that vitamin B12 may hold promise as a PD therapy.
Parkinsons disease, the most common chronic neurodegenerative disorder, affects one percent of the world population over the age of 60 by disabling the brain and disrupting both motor and cognitive function. Missense mutations in the gene leucine-rich repeat kinase 2 are the greatest known genetic contributor to PD and are linked to the incidence of both familial and sporadic forms of the disease. These mutations lead to a hyperactive form of the protein kinase that promotes neurotoxicity. Over time, increases in LRRK2 kinase activity lead to a reduction in the activity of dopamine in the brain, which manifests in the muscle rigidity and tremors that are hallmark symptoms of PD. Over the past several years, drug companies have developed LRRK2 kinase inhibitors that target the adenosine triphosphate binding pocketessentially, the energy storein the biological system of LRRK2, which is required for LRRK2 signaling.
The Role Of Cholinergic Dysfunction In Nonmotor Symptoms Of Pd
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B12 And Parkinsons Disease
This study included 680 patients over a two year period. It included individuals who had been diagnosed with early Parkinsons disease however, they had not begun medication at this time. The research team assessed serum B12, serum MMA, homocysteine, and holotranscobalamin. These were measured at baseline and every three months up to two years. Outcomes assessments included the unified Parkinsons disease rating scale , ambulatory capacity score, and mini-mental state examination .
Elevated homocysteine levels at baseline were associated with worse scores on the MMSE, which is associated with greater cognitive decline. The patients with low B12 status developed greater morbidity as assessed by greater worsening of the ambulatory capacity score.
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Animals And Brain Slice Preparation
The use of the animals followed the National Institutes of Health guidelines and was approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University. All efforts were made to minimize the number of animals used. BAC LRRK2 transgenic mice were obtained from Chenjian Lis laboratory at Weill Medical College of Cornell University and maintained on Taconic FVB/N background and BAC LRRK2 TG mice previously described maintained on C57/NJ background.
Three- to 5-month-old male transgenic LRRK2-G2019S mice and their non-transgenic littermates were used for LRRK2 kinase inhibition in striatal brain slices. For preparing striatal slices, mice were decapitated without anesthesia after cervical dislocation and brains were immediately dissected out. Coronal striatal brain slices at 250m were prepared on a vibratome . The striatal slices were allowed to recover for 0.51h at 36°C in a holding chamber containing oxygenated artificial CSF . To examine the effects of the LRRK2 inhibitors, slices were incubated for 2h in oxygenated ACSF containing LRRK2 inhibitors. For the incubation treatment, striatal slices were bisected, and one striatum was exposed to LRRK2 inhibitor while the other was exposed to vehicle . After treatment, the slices were collected and rapidly frozen in dry ice and stored in 80°C until assayed.
Identification Of Vitamin B12 As A Lrrk2 Kinase Activity Inhibitor
AdoCbl was identified as a kinase activity inhibitor of FLAG-tagged wild-type LRRK2 purified from BAC transgenic mouse brain from a high-throughput screen of a small library of 2,080 FDA-approved compounds . To assay LRRK2 kinase activity, we measured the time-resolved fluorescence resonance energy transfer between phosphorylated Fluorescein-LRRKtide peptide and Terbidium-labeled anti-pLRRKtide antibody. In this screen AdoCbl displayed a half-maximal inhibitory concentration of 1.2M . Vitamin B12 consists of a central cobalt ion that is equatorially chelated by a tetradentate corrin macrocycle and up to two axially-coordinating ligands . The lower -coordinating ligand is usually a dimethylbenzimidazole base that connects to the f-side chain of the chelator by an -ribazole-containing backbone.
Vitamin B12 has additional forms in addition to AdoCbl, including cyanocobalamin , hydroxycobalamin , and methylcobalamin , which are distinguished by their -coordinating ligand . Only MeCbl and AdoCbl are physiologically active in cells, as coenzymes of MeCbl-dependent methionine synthase and AdoCbl-dependent methylmalonyl coenzyme A mutase. In human, these enzymatic reactions play a key role in the metabolism of amino acids, nucleotides, and fatty acids, in addition to their normal function in the nervous system, and the formation of red blood cells.
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Folic Acid And Vitamin B12 Level
Fasting venous blood was obtained from all subjects in the morning. The serum was then separated by centrifugation in the Hospital’s biochemistry laboratory to allow vitamin B12 and folic acid levels to be assessed. Specifically, vitamin B12 was measured using a microparticle enzyme immunoassay performed using the AxSYM automatic immune analyzer . In contrast, serum folic acid levels were measured using an ELISA kit . Absorbances were read using a microplate reader . In accordance with Nanjing Brain Hospital’s standards, normal range of vitamin B12was defined as being between 189 and 883 pg/ml, whereas the normal range of folic acid was defined as being between 1.1 and 20.5 ng/ml.
In Vitro Kinase Assays
Kinase reactions were performed in 30µL kinase buffer at 37°C for 30min in the presence of ATP and 50µM cold ATP. Beforehand, LRRK2 or Roco4 Kinase was loaded with inhibitor and/or substrate, followed by 30min incubation on ice. Reactions were stopped by addition of Laemmli buffer and boiling at 95°C for 10min. Samples were resolved on 412% SDS-PAGE pre-cast gels . Radioactive signal was captured onto a phosphor-screen and was digitally collected using a Typhoon scanner. ImageQuant densitometry was used to quantify the phosphor-signal.
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Vitamins For Parkinson’s Disease
Verywell / Michela Buttignol
Apart from traditional pharmaceutical treatments, if you have Parkinsons disease, your doctor may recommend vitamins with antioxidant properties. While it is best to get these from food sources as part of a healthy, balanced diet, some people need to take supplements. These vitamins include:
- Vitamin B12
Its Not Necessarily Alzheimers
More than 50 conditions can cause or mimic the symptoms of dementia, and a small percentage of dementias are reversible. Two common examples are dementia caused by vitamin B12 deficiency or an underactive thyroid . Getting the right diagnosis is important so that you know what options you have, because symptoms subside when the underlying problem is treated.
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How About The Motor Symptoms
Its not yet clear whether its consumption can also treat the typical motor symptoms of Parkinsons. So far only one study has reported that it may help improve balance in patients who are younger. In this study, a high dose of vitamin D supplement for 16 weeks was found to cause a significant improvement in the balance of those patients who were 66 or younger, compared with the placebo group or those patients who were 67 and older.
Low Vitamin B12 And Parkinson Disease
- Stuart J. McCarterCorrespondenceCorrespondence: Address to Stuart J. McCarter, MD, Mayo Clinic Department of Neurology, 200 First Street SW, Rochester, MN 55905.Mayo Clinic Department of Neurology, Rochester, MNMayo Clinic Department of Sleep Medicine, Rochester, MN
- Mayo Clinic Department of Nutrition, Rochester, MNDepartment of Medicine, Minneapolis, MN
- Mayo Clinic Department of Sleep Medicine, Rochester, MNUniversity of Minnesota Medical School, Minneapolis, MN
- Mayo Clinic Department of Neurology, Rochester, MNMayo Clinic Department of Sleep Medicine, Rochester, MNMayo Clinic Department of Medicine, Rochester, MN
- Mayo Clinic Department of Neurology, Rochester, MNMayo Clinic Department of Health Sciences Research, Rochester, MN
Curr Neurol Neurosci Rep.
Curr Neurol Neurosci Rep.Mov Disord.Nutrients.Mov Disord.
What Causes Parkinsons Disease
Parkinsons is caused by the degeneration of brain cells in an area of the brain called the substantia nigra. These neurons are responsible for the production of a particular neurotransmitter called dopamine and it is the lack of this neurotransmitter that is responsible for the main Parkinsons symptoms. The cause of the disease is not known. However, like most degenerative illnesses, it is likely to be due to a range of factors including interactions between genes and environment. Contributory factors may include environmental toxicity, physical trauma, genetics, drugs, disease , nutritional deficiency, mitochondrial insufficiency, enzyme deficiency and unremitting stress.
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Many Peopleespecially The Elderlyare Deficient In Vitamin B12
Many people are deficient in vitamin B12, and other B vitamins, for multiple reasons.
For instance, some circumstances either boost the bodys demand for B vitamins or greatly inhibit vitamin B absorption, making supplementation necessary.
Individuals in the following categories have the greatest risk of a vitamin B12 deficiency. For these individuals, the need for a daily supplement can be much more critical.
Many older adults experience a decline in their appetite, reducing their overall dietary intake of all B vitamins. Older individuals may also be unable to absorb naturally occurring vitamin B12. With age, many people develop a loss of certain important functions of the stomach and digestive tract. This includes a decreased production of stomach acid, an intrinsic factor needed to release B12 from foods, and for its absorption in the small intestine.31,32
Commonly prescribed drugs that reduce stomach acid production decrease absorption of vitamin B12.33 Metformin, the popular diabetes drug, is known to interfere with the absorption of vitamin B12.34,35 Birth control pills can also deplete B12 and other B vitamins.36
B vitamins, especially B12, are important for healthy fetal development. In breast-feeding or pregnant women, a deficiency of B12 can result in severe neurological damage or birth defects in the infant or fetus.37,38
Some Medical Conditions
Vegetarians and Vegans
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Parkinsons And Weight Gain
Parkinsons medication does not tend to make people gain weight, but a small number of people may experience impulsive and compulsive behaviour. This is a side effect of some Parkinsons medication, particularly dopamine agonists and, in some cases, levodopa.
Impulsive behaviour is when a person cant resist the temptation to carry out certain activities. These are often activities that give an immediate reward or pleasure, such as gambling, hypersexuality and overeating.
So, someone may eat large amounts of food in a short period of time because they cant control their appetite, and as a result, they gain weight.
If you think youre experiencing this behaviour, speak to your GP, specialist or Parkinsons nurse.
We dont advise anyone to stop taking or to change their Parkinsons medication without the advice of their specialist or Parkinsons nurse.
Any changes have to be made slowly and gradually, and should always be carried out and reviewed by a specialist, because of the risk of side effects and withdrawal symptoms.
Someone experiencing impulsive or compulsive behaviour may not realise they have a problem. So its important that their carer is aware of these side effects.
Deep brain stimulation and weight gain
Some people with Parkinsons may put on weight quickly after having deep brain stimulation, a surgery sometimes used to treat the condition.
If you think youre experiencing this behaviour, speak to your GP, specialist or Parkinsons nurse.