<u>NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration</u> Nature, Published online 16 March 2008
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature06721.html

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration1, 2. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.

Hi Barbara and Dan,

This is very interesting. It leaves me asking questions though. Inhibition of PARP leaves extra NAD to fuel NADPH oxidase. In view of the fact that the NADPH oxidase inhibitor Apocynin greatly increased lifespan in the mice has me wonder what will happen when you fuel NADPH oxidase with nicotinamide. Increased PARP is reported in the brain in ALS, yet reduced PARP is reported in the large motor neurons of SALS. I'd wondered whether apocynin may indirectly increase PARP in the large motor neurons. You'd mentioned in your previous post that nicotinamide may increase PARP due to the fact that it makes NAD available. It seems that apocynin may do the same.

Mary

Hi Barbara,

I'd suggest the opposite. That's why I ask so many questions. I haven't actually met anyone in person with ALS. Just a reminder that I'm a ex-nurse who joined this list to find out about SOD1 as I have a child with Down syndrome. I suppose I should be trying to influence DS researchers to look at ATN-224.

Mary

Barbarawanda,

I am concerned with using high amounts of niacinamide due to the sensitivity that slow acetylators have with the aromatic ring of the niacinamide molecule. This is similar to the reactions seen with isoniazid used to treat tuberculosis. Some patients develop peripheral neuritis accompanied by high antinuclear antibodies associated with drug induced lupus. Because some ALS patients have lupus antibodies the connection was too close for comfort.

I looked up some data on ALS detoxification pathways. I thought perhaps ALS would lean one way or the other. Fifty percent of the US population are slow acetylators. According to the enclosed document this 1:1 ratio between slow and fast acetylators is similarly found in ALS. (1)

Slow acetylators will dump Vitamin B-6 into the urine in the presence of excess niacinamide, isoniazid, etc. causing a secondary B-6 deficiency. By supplying B-6 in conjunction with niacinamide all the symptoms disappear. Any reaction to using too much B-6 can be eliminated with magnesium.

Each one of these genetically conferred liver detoxification pathways has its own set of problems with intermediate metabolites. Fast acetylation is associated with large intestine cancer. Slow acetylation is associated with prostate, breast, and thyroid cancer.

Acetylation speed may interfere with half of your test results without addition of B-6 and magnesium.

The other concern I have with niacinamide is the cholesterol lowering effect. High cholesterol is found to be predictive of increased survival in ALS.(2)

Probably due to the cholesterol lowering effect, many reputable vitamin companies are putting high amounts of niacin and/or niacinamide relative to the amount of B6 in their B Complex formulas. It should no less than a 1:1 ratio. I would prefer a higher amount of B6 than niacin because B-6 along with magnesium drives metabolic pathways more often than anything else.

Best of luck,

Donna

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References:

(1)HGM2003 Workshop Abstracts
2. Medical Genomics I

Presentation 64
Molecular genetic analysis of sporadic amyotrophic lateral sclerosis patients from Russia
1M.I. Shadrina, 1P.A. Slominsky, 1E.A. Kondratyeva, 1A.L. Zherebtsova, 2A. Alekhin, 2V.I. Skvortsova, 1S.A. Limborska
1Institute of Molecular Genetics, Russian Scientific Academy, Kurchatov sq.2, Moscow, Russia, 123182, 2Department of Fundamental and Clinical Neurology, Russian State Medical University, Lenskay st.15, Moscow, Russia 129327

At present the cause of amyotrophic lateral sclerosis (ALS) remains uknown. The majority of ALS cases (90%) are sporadic and 10% of ALS patients are familial cases (FALS). The clinical and pathological characteristics of FALS and sporadic ALS (SALS) are almost identical. Current evidence suggests that genetic factors triggering focal initiation and then spreading of motor neuron degeneration may be implicated in ALS pathogenesis. Various hypotheses have been suggested as potential contributors of disease such as oxidative damage, detoxification processes, cytoskeletal damage and excitotoxicity. Genes involved in these processes, such as CuZn-superoxidedismutase gene (SOD1), Mn-superoxidedismutase gene (SOD2), glutathione S-transferase genes (GSTT1 and GSTM1), N-acetyltrasferase gene (NAT2), heavy subunit of neurofilaments gene (NEFH), glutamate transporter gene (SLC1A2) can play a causal role in ALS development. To investigate a role of these genes in ALS, we study mutations and polymorphisms in these genes in 51 patients with SALS in Moscow and control unrelated population from Russia. The mutation analysis of SOD1 has revealed two patients (4%) with D90A mutation and one patient (2%) with G12R mutation. The screening for A9V polymorphism of SOD2 gene has no significant differences between frequency of A9A, A9V and V9V genotypes in patients (21,6%, 49% and 29,4% respectively) and controls (18%, 54%, 28%). We found no difference between different genotype variants for GSTM1 and GSTT1 loci in our samples, including null genotype (0/0), which determines low level of enzyme activities and detoxification (GSTT1(0/0) genotype: 12% in controls and 21% in patients (c2=1,56; P&lt;0,25); GSTM1 (0/0) genotype: 24% in controls and 38% in patients (c2=2,59; P&lt;0,2).

***The comparative analysis for FF genotype (fast acetylators) and FS+SS genotype (slow acetylators) in NAT2 gene has not revealed any distinction in frequencies of these genotypes between patients (FF-51,92%, FS+SS-48,07%) and controls (FF-52%, FS+SS-48%)***.

Study of G603A substitution polymorphism in exon 5 of the SLC1A2 gene also has not found also differences between frequency of G603 and A603 alleles in patients (33% and 67% respectively) and controls (25% and 75%). Only when we have investigated L/S polymorphism of NEFH gene, we have found significant differences in SS genotype frequency between controls (6,7%) and patients (32,7%) (X2=9.97, p&lt; 0.005). We conclude that SS genotype of NEFH gene, probably, is associated with the pathogenesis of ALS.

Generated by AbProcess V2.10 - Copyright © 1999-2003 Alastair Brown

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(2)Neurology. 2008 Jan 16; : 18199832

Dyslipidemia is a protective factor in amyotrophic lateral sclerosis.

L Dupuis , P Corcia , A Fergani , J-L Gonzalez De Aguilar , D Bonnefont-Roussel , R Bittar , D Seilhean , J-J Hauw , L Lacomblez , J-P Loeffler , V Meininger

ABSTRACT BACKGROUND: Amyotrophic lateral sclerosis (ALS) is the most serious form of degenerative motor neuron disease in adults, characterized by upper and lower motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. High prevalence of malnutrition and weight loss adversely affect quality of life. Moreover, two thirds of patients develop a hypermetabolism of unknown cause, leading to increased resting energy expenditure. Inasmuch as lipids are the major source of energy for muscles, we determined the status of lipids in a population of patients with ALS and investigated whether lipid contents may have an impact on disease progression and survival. METHODS: Blood concentrations of triglycerides, cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured in a cohort of 369 patients with ALS and compared to a control group of 286 healthy subjects. Postmortem histologic examination was performed on liver specimens from 59 other patients with ALS and 16 patients with Parkinson disease (PD). RESULTS: The frequency of hyperlipidemia, as revealed by increased plasma levels of total cholesterol or LDL, was twofold higher in patients with ALS than in control subjects. As a result, steatosis of the liver was more pronounced in patients with ALS than in patients with PD. Correlation studies demonstrated that bearing an abnormally elevated LDL/HDL ratio significantly increased survival by more than 12 months. CONCLUSIONS: Hyperlipidemia is a significant prognostic factor for survival of patients with amyotrophic lateral sclerosis. This finding highlights the importance of nutritional intervention strategies on disease progression and claims our attention when treating these patients with lipid-lowering drugs.

NIACIN NUTRITION AND MODULATION OF NAD LEVELS

First I need to clarify:- Vitamin B3 is niacin is aka nictoinamide / nicotinic acid - either of which can be used to make NAD in humans. The paper below (the only one I can find which has assessed this) demonstrates that a dietary supplement of 100 mg / day of nicotinic acid raises NAD levels in healthy volunteers up to 5-fold - a remarkable result, and what we would hope would exert neuroprotective effects. Furthermore, interchanging nicotinamide for nicotinic acid would presumptively remove the high cholesterol caveat raised by Donna. I'll post later on on the obverse experiments - showing that even marginal deficits in niacin nutrition can markedly reduce NAD levels in healthy volunteers. I am away on holiday in a few days time so won't be able to reply to further queries for a week
Barbarawanda


Authors Full Name Weitberg, A B.

Institution Division of Hematology/Oncology, Roger Williams Cancer Center, Providence, RI 02908.

Title Effect of nicotinic acid supplementation in vivo on oxygen radical-induced genetic damage in human lymphocytes.

Source Mutation Research. 216(4):197-201, 1989 Aug.

Abstract The ability of nicotinic acid to protect human lymphocytes in vivo against oxygen radical-induced DNA strand breakage was tested. NAD+ concentrations rose progressively in lymphocytes to nearly 5 times baseline levels in human volunteers ingesting nicotinic acid (100 mg/day) for 8 weeks. Strand breaks decreased proportionately to NAD+ concentrations over this time period in lymphocytes exposed to oxygen radicals. After 8 weeks of supplementation with nicotinic acid, radical-treated lymphocytes incubated for 24 h evidenced significantly less DNA damage compared to controls.

Publication Type Journal Article. Research Support, Non-U.S. Gov't. Research Support, U.S. Gov't, P.H.S..

Hi Joanna,

Sorry, I don't know. I would like to see studies with both PARP inhibitors as well as nicotinamide though, as it may give us some clues.

Something else to consider is that interferon gamma drives the tryptophan - quinolinic acid breakdown. Increased quinolinic acid is reported in ALS. NAD is produced downstream of quinolinic acid. Is there a failure there or is there also increased NAD. That may explain increased PARP and NADPH oxidase in some regions.

If there is increased tryptophan breakdown due to high interferon gamma levels, the maybe Anti-interferon from Dr Maroun's lab is worth a try.

Mary

De novo production

Metabolic pathways that synthesize and consume NAD+. The abbreviations are defined in the text.Most organisms can synthesize NAD(P) from simple components. The exact set of reactions differ among organsisms, but a common feature is the generation of quinolinic acid (QA) from an amino acid - either tryptophan (Trp) in animals and some bacteria, or aspartic acid in some bacteria and plants.[1] The quinolinic acid is converted to nicotinic acid mononucleotide (NaMN) by transfer of a phosphoribose moiety. An adenyl group is then transferred to form nicotinic acid adenine dinucleotide (NaAD). Finally, the nicotinic acid group in NaAD is amidated to an nicotinamide (Nam) group, forming nicotinamide adenine dinucleotide.[2]

In a further step, some NAD+ is converted into NADP+ by [[NAD+ kinase]], which phosphorylates NAD+.[3] In most organisms, this enzyme uses ATP as the source of the phosphate group, while in some bacteria such as Mycobacterium tuberculosis and archaea such as Pyrococcus horikoshii, inorganic polyphosphate acts as an alternative phosphate donor.[4][5]

Neurodegener Dis. 2005;2(3-4):166-76. Links
Implications for the kynurenine pathway and quinolinic acid in amyotrophic lateral sclerosis.Guillemin GJ, Meininger V, Brew BJ.
Centre for Immunology and University of New South Wales, School of Medicine and School of Medical Sciences, Sydney, Australia. g.guillemin@cfi.unsw.edu.au

The kynurenine pathway (KP) is a major route of L-tryptophan catabolism leading to production of several neurobiologically active molecules. Among them is the excitotoxin quinolinic acid (QUIN) that is known to be involved in the pathogenesis of several major inflammatory neurological diseases. In amyotrophic lateral sclerosis (ALS) degeneration of motor neurons is associated with a chronic and local inflammation (presence of activated microglia and astrocytes). There is emerging evidence that the KP is important in ALS. Recently, we demonstrated that QUIN is significantly increased in serum and CSF of ALS patients. Moreover, most of the factors associated with QUIN toxicity are found in ALS, implying that QUIN may play a substantial role in the neuropathogenesis of ALS. This review details the potential role the KP has in ALS and advances a testable hypothetical model.

PMID: 16909022 [PubMed - indexed for MEDLINE]

Hi Barbara,

I'd questioned whether we may expect increased NAD and nicotinamide levels in ALS as quinolinic acid levels have been found to be increased. If so, does that increase PARP and NOX2 expression?

I've been looking at this Kegg chart and the enzymes expressed in humans are green. It seems that NAD and nicotinamide are metabolized from quinolinic acid in humans, although, as you suggest, it may not be the major source. I guess it must play some part.

Nicotinamide: http://www.genome.ad.jp/dbget-bin/get_pathway?org_name=hsa&mapno=00760

Mary

I'm surfing medline today! Here's another more recent study demonstrating that nicotinic acid can raise cellular NAD levels
Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells.

Authors Full Name Hara, Nobumasa. Yamada, Kazuo. Shibata, Tomoko. Osago, Harumi. Hashimoto, Tatsuya. Tsuchiya, Mikako.

Institution Department of Biochemistry, Shimane University Faculty of Medicine, 89-1, Izumo, Shimane 693-8501, Japan. nhara@shimane-u.ac.jp

Title Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells.

Source Journal of Biological Chemistry. 282(34):24574-82, 2007 Aug 24.

Abstract NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated [(14)C]NAD synthesis from [(14)C]NA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H(2)O(2). Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools.

Publication Type Journal Article.

Hi Barabara, When the Engelhardt team discovered a ROS cycle in the SOD1 mouse, I assumed that there might be a ROS cycle, or ROS cycles, in sporadic ALS. Thanks for articulating one.

I was wondering about the use of PARP inhibitors alone though. What would happen to cell with damaged DNA if they do not have PARP to either protect them or put the cells into apoptosis. In other words, what happens to cells that have damaged DNA, but are not 'fixed' by PARP?

Maybe, as you say, a combination of PARP inhibitors with nicotinic acid would be better?

Or maybe nicotinic acid by itself? (Although I think a combination might be better.)

Hi Joanna
I seem to have posted my model twice! (I thought I wasn't successful first time.

Anyway, a perceptive questiom regarding PARP-1's known role in DNA repair - my expertise as it happens (for a change - I seem to spend my time on this site pontificating about subjects where I am acutely aware I should be better informed). What happens if you inhibit PARP-1, DNA repair slows dowm - but it is not complete;y prevented. Now in proliferating cells, this slowing down of repair can result in damaged DNA entering the replication fork during DNA synthesis - with lethal consequences. In marked contrast, non-proliferating cells like motor neurones can readily survive this slowing of repair since no DNA synthesis on a damaged template occurs. In this case, although DNA damage is occurring due to excessive ROS, the danger to the motor neurone is not the unrepaired DNA damage per se, but the ensuing constitutive activation of PARP-1 leading to serious NAD depletion etc, as outlined in my model.

Cheers

Barbara