Amyotrophic Lateral Sclerosis.
Motoneuron Diseases

CHIARA BACCINO

GIULIA CATOZZI

PESTICIDE EXPOSURE IN SPORADIC ALS RISK

Introduction

Amyotrophic lateral sclerosis is a degenerative disorder, typically fatal within five years. It’s a late onset disease in which motor neurons deteriorate leading to muscular atrophy, weakness, and death due to respiratory failure.

The definite aetiology of ALS has not been detected. About 10% of cases are inherited as an autosomal dominant trait (familial ALS); in contrast 90% of ALS cases are sporadic in nature (sporadic ALS). This inequality indicates there is likely to be a significant contribution of non-genetic factors, such as exposure to adverse environmental agents, including heavy metal and pesticides.
Pesticides are strong candidates to be examined for their risk of inducing ALS. Some epidemiological and animal studies have considered the relationship between exposure to pesticides, especially organophosphates, and the risk of developing ALS, with significant results.

The role of environmental mercury, lead and pesticide exposure in development of ALS, 2009
Association of pesticide exposure with neurologic dysfunction and disease, 2004
Familial, environmental and occupational risk factors in development of als, 2012

Organophosphates

The Organophosphates are esters of phosphoric acid or phosphorothioic acid, and probably the most pervasive organophosphorus compounds.
Organophosphorus pesticides were synthetized for the first time in 1937, but many of them showed a strong toxicity and were then developed as chemical weapons, principally as nerve gas. Even though the actual pesticides are less toxic, they derive as well from nerve gas compounds, such as sarin, soman, tabun. The major components in the actual OP pesticides are parathion and the oxygenate form paraoxon, both extremely toxic.

OPs Toxicity

OPs exert their toxicity inhibiting acetylcholinesterase enzyme, responsible for acetylcholine degradation. The reaction between organophosphate compounds and the active site of acetylcholinesterase (an hydroxyl group upon a Serine) lead to forming a transitory intermediate complex then hydrolyzed which loses its aryl or alkyl group resulting in the non-reactive, phosphorylated enzyme, stable and almost irreversibly inhibited.

The non-degraded acetylcholine accumulates in the intersinaptic area, constantly stimulating its receptors.
Intoxication symptoms derive by stimulation of muscarinic and nicotinic receptors in both sympathetic and parasympathetic nervous system and in neuromuscular junctions.

OPs were demonstrated to give persistent neurotoxicity and several syndrome were discovered, due to exposure to very high pesticides doses, e.g. in case of accidents or suicide attempts by pesticides ingestion in rural areas. Among them, Ops were discovered to be responsible for the Organo Phosphate Induced Delayed Neuropathy in which there are several signs similar to ALS symptoms, such as spasticity, hyperreflexia, muscular weakness and flaccidity, muscle twitches, especially subsequently to a primary reflex reduction (polyneuropathic ALS), indicating a permanent injury to the superior motor neuron as in ALS. Although correlation between pesticides and ALS has not been scientifically demonstrated yet, the evidence of other organophosphate induced syndromes with ALS-like symptoms could make the correlation hypothesis stronger.

Absorption and biotransformation

The reason why OPs toxicity appears only for high doses exposure is that, for under threshold levels, several mechanism perform a large biotransformation process. Most of OPs compounds are absorbed from skin, conjunctiva, gastrointestinal tract and lung. After absorption, biotransformation process includes two phases: phase one enzymes are oxidative, reductive or hydrolytic enzymes, while phase two enzymes catalyse transfer or conjugation reaction of phase one pathways products with glutathione, glucuronic acid, glycine etc. making them easier to eliminate.

Organophosphates includes both phosphoric acid esters and phosphorothioic acid esters, such as Parathion, which is immediately desulfurized and oxygenated in Paraoxon with reactions involving cytochrome P 450. Then Paraoxon, or desulfurized Parathion, is hydrolyzed, mainly by PARAOXONASE 1.

PON1 reaction

PON1 Gene

The human paraoxonase gene cluster is located on chromosome 7q21.3. The cluster comprises three genes ordered PON1, PON3 and PON2. The PON1 enzyme acts as the major protective mechanism by which toxic exogenous compounds are hydrolyzed in serum. It’s also known that PON1 has two non-synonymous coding SNPs and a promoter SNP which alter enzyme activity affecting expression levels and impairing its detoxifying activity. It’s in this cases, that is to say in genetically predisposed host, in which the body concentration of PON1 is suboptimal, intrinsic OPs toxicity is not prevented and exposure to environmental toxins may precipitate motor neurons degeneration.

The increased susceptibility to OPs may increase the risk of sALS, making motor neuron degenerate by overstimulation caused by anticholinesterasic OPs activity.

PON1 ALS-Related Polymorphism

An increased risk of ALS among people with PON1 polymorphisms that lead to reduced OP compounds detoxification capability is even suggested by several reports that postulate that the risk of developing ALS is bigger if there is an increase in mutation of PON1 gene and if the serum concentration of PON1 is suboptimal.

Several PON1 variants in the coding and promoter region determine the catalytic activity and the enzyme expression level.

A glutamine/arginine (Q/R) polymorphism at amino acid position 192 has been described. PON1R192 allele is associated with higher serum paraoxonase activity, compared to PON1Q192. The Arginine isoform hydrolyzes paraoxon at a high rate, while the Glutamine isoform works at a low rate. Several studies have confirmed that the prevalence of chronic toxicity increase in a stepwise fashion from individuals with a “fast metabolism” genotype ( R) to individuals with a “slow metabolism” genotype (Q), demonstrating the PON1 genotype is an important determinant of susceptibility to pesticide poisoning.

A second coding polymorphism is determined by a Leucine/Methionine substitution at position 55 (L55M). A lower serum concentration level of allozyme has been found in individuals with Methionine allele. The Leucine genotype is supposed to confer a better protection against Ops toxicity, since several studies report that PON155M was overrepresented in sALS vs controls. However, this appears to be due to linkage disequilibrium with a polymorphism in the 5’ regulatory region of PON1 (C108T). The C108T polymorphism has, among other SNPs identified in this region, the greatest effect on plasma PON1 levels, with the -108C allele producing about twice level of plasma PON1 as the -108T allele.

A large-scale international meta-analysis of paraoxonase gene polymorphisms in sporadic ALS, 2009
Paraoxonase (PON1) polymorphism and activity as the determinants of sensitivity to organophosphates in human subjects, 2007
Association between human paraoxonase gene polymorphism and chronic symptoms in pesticide-exposed workers, 2003
Interaction between PON1 and population density in amyotrophic lateral sclerosis, 2009

Conclusions

The PON1 SNPs have relatively high frequencies. Association of common variations in PON1 with sALS and other neurodegenerative diseases may have important implications. If environmental substrates, such as pesticides, could increase risk of neurodegeneration for a significant genetically predisposed rate of population, it follows that occupational health strategies to minimise the exposure should be prioritised. Additionally, promoting further studies may help in exploring paraoxonases therapeutic potential.

FDA Finds No Link Between Statin Use and ALS

Michael O'Riordan

Medscape Medical News 2008. © 2008 Medscape

October 2, 2008 (Silver Springs, MD) — A new Food and Drug Administration analysis has shown that statins do not increase the risk of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease [1].

The meta-analysis, which included 41 clinical trials and was published online September 29, 2008 in the journal of Pharmacoepidemiology and Drug Safety, revealed similar rates of ALS in the statin-treated and placebo-treated patients, but the agency says that caution is still needed because of the expansive use of the drugs and the dire consequences of this neurodegenerative disorder.

"People should look at this paper as one bit of evidence," said lead investigator Dr Eric Colman (Food and Drug Administration, Silver Spring, MD). "You have to be a little cautious about how much you make of the results. We just look at this as one bit of information in a much larger puzzle, and we're going to have to see what other data look like."

Speaking with heartwire, Colman said the agency observed a disproportionate number of reports for ALS in association with statin use in the adverse-event databases. In 2006, there were 91 reports of ALS with statins in the agency's adverse-event reporting system (AERS), and these findings prompted internal discussions that eventually led to the meta-analysis.

"It got a point where we thought it would be useful to go back to look at the clinical-trials data to see what kind of numbers there were for ALS in those trials," said Colman. "It's often tough to know what to make of data-mining signals. You really can say very little about most of them, but this was a spark for us to go back and look at the trial data."

The statin manufacturers were requested by the agency to provide data from placebo-controlled trials and the occurrence of ALS in patients during the study or poststudy follow-up. The 41 clinical trials ranged in duration from six months to five years and represented approximately 200 000 patient-years of exposure to statin therapy and 200 000 patient-years of exposure to placebo. Overall, the incidence of ALS was similar in the statin-treated and placebo-treated patients.

Cases of ALS From Clinical Studies of Statins
Therapy Patients, n Person-years ALS cases Incidence/100 000 person-years
Statins 64 602 212 775 9 4.2

Colman said the incidence of ALS in the general population is one to two cases per 100 000 person-years, and the incidence increases with age. He said he is unaware of any biological mechanism whereby statins would cause ALS, but, as has been reported by others, statins might unmask or alter the clinical course of disease.

The agency points out that none of the clinical trials were designed to examine the incidence of ALS, and in the absence of formal adjudication, some of ALS cases might have been misdiagnosed. Also, follow-up after the completion of the studies was short, usually only 30 days, and might lead to some ALS cases being missed.

Colman said there is an ongoing case-control study by Dr Lorene Nelson at Stanford University investigating the possible link between statin therapy and ALS risk. The agency also stressed that the benefits of statins outweigh the risks in individuals with or at high risk for cardiovascular disease.

1. Colman E, Szarfman A, Wyeth J, et al. An evaluation of a data mining signal for amyotrophic lateral sclerosis and statins detected in FDA's spontaneous adverse event reporting system. Pharmacoepidemiol Drug Saf 2008; DOI: 10.1002/pds.1643. Available at. 18821724

J Neurol Sci. 1986 Jan;72(1):49-60.
"Collagenase activity in skin fibroblasts of patients with amyotrophic lateral sclerosis.":

Beach RL, Rao JS, Festoff BW, Reyes ET, Yanagihara R, Gajdusek DC.
Abstract

In the current study we have measured collagenase activity released from skin explants and fibroblasts of patients with both Guam-type and sporadic amyotrophic lateral sclerosis and controls. The rationale for such a study derives from work reported more than 20 years ago demonstrating abnormalities in skin collagen metabolism in patients with the disease. We were not able to find significant differences in collagenase activity when fibroblasts were compared relative to the total protein secreted. This is explained, in part, by our finding of an increase in total protein released from fibroblasts of the amyotrophic lateral sclerosis patient group. Increased collagenase release did occur when activity was expressed per number of cells plated but was not statistically significant. In addition, increased release followed a 3-day lag period in skin organ culture. These results suggest that collagenase and other enzymes known to activate collagenase, such as plasminogen activator, capable of degrading extracellular matrix components might be responsible for the increased collagenolytic activity previously observed in amyotrophic lateral sclerosis patients' skin. Further evaluation of extracellular-acting degradative enzymes from skin, muscle, nerve and central nervous system may be important to follow-up such leads in understanding the pathogenesis of this enigmatic and fatal disorder.

MECCANISMI MOLECOLARI COINVOLTI NELLA DEGENERAZIONE DEL MOTONEURONE NEI PAZIENTI AFFETTI DA SCLEROSI LATERALE AMIOTROFICA MUTANTI PER IL GENE SOD1

DEFINITION

La Sclerosi laterale amiotrofica (SLA) detta anche morbo di Lou Gehrig o mallatia di Charcot e’ una malattia degenerativa e progressiva che colpisce i motoneuroni sia a livello corticale(1° motoneurone) che a livello del tronco encefalico e del midollo spinale(2°motoneurone).

EPIDEMIOLOGY

E’ una patologia che si presenta con un incidenza di 2 casi/100.000/anno.In genere colpisce soggetti compresi tra i 40 e 70 anni con un rapporto nel uomo e nella donna di 2:1.Nella maggioranza dei casi si presenta in forma sporadica ma ne esistono anche forme familiari che si presentano nel 5% dei pazienti.

SYMPTOMS

Possiamo distinguere la SLA in tre forme:

La forma COMUNE si manifesta con debutto insidioso e progressivo di riduzione di forza alle mani e poi progressiva atrofia dell’arto fino a coinvolgere i muscoli della spalla.
Il deficit e’ tipicamente simmetrico seppur puo’ essere laterale per lungo tempo.Le fascicolazioni sono un reperto caratteristico della malattia.
La compromissione della via cortico spinale si manifesta con iperreflessia dei 4 arti.
In fase avanzate il segno di Babinsky e’ positivo in seguito all’ipertonia piramidale.Frequenti sono anche le turbe vasomotorie(spasmo vasogenico).

La forma BULBARE ha un esordio con deficit di pronuncia dei fonemi consonantici,ipotrofia e fascicolazioni ai margini linguali,difficolta e poi impossibilita’di protrusione linguale.
In fasi successive si ha una disfagia lusoria(prima per i liquidi),disfonia da ipotonia laringea,dispnea inspiratoria.I deficit del VII portano ad una riduzione della mimica,difficilta’ a chiudere le labbra e scolo di saliva.

Nella forma POLINEUROPATICA e’ presente un deficit motorio ai muscoli della loggia anteriore esterna della gamba:il soggetto tende ad inciampare per la caduta del piede o ha difficolta’ a sollevarsi dalla posizione seduta.I crampi sono molto comuni cosi come le fascicolazioni.In genere questi disturbi si presenta prima dell’atrofia.In un primo tempo c’e’ una riduzione dei riflessi profondi mentre i segni piramidali compaiono successivamente.

DIAGNOSIS

I criteri oggetivi sono:
-assenza di disturbi della sensibilita’
-assenza di disturbi sfinterici
Il segno di Babinsky positivo evidenzia il danno al 1° motoneurone invece il danno al 2° motoneurone si manifesta con debolezza,atrofia muscolare,crampi muscolari e fascicolazioni che rappresentano un criterio importante.
L’elettromiografia (EMG) da diverse indicazioni quali:
-ridotto numero di potenziali dell’unita’ motoria
-aumento della durata e dell’ampiezza media
-potenziali di fibrillazione
-riduzione della velocita di conduzione del 10% senza blocchi di conduzione,solo su nervi motori.
Con l’EMG si effetua un’accurata diagnosi differenziale con alcune miopatie.
La RMN spesso negativa in questi pazienti puo’ escludere altre patologie con sintomatologia simile(sclerosi multipla,siringomielia).
In alcuni pazienti sono documentate alterazioni dei potenziali motori evocati (PEM) da stimolazione magnetica transcranica,con compromissione della via cortico spinale.

PATHOGENESIS

Nellla SLA si osservano alcuni aspetti caratteristici:
-La perdita dei motoneuroni delle corna anteriori del midollo spinale,della corteccia e del tronco encefalico
-Degenerazione del fascio cortico spinale

I muscoli presentano il quadro dell’atrofia neurogena con fibre normali alternate a fibre in degenerazioni.
Gli aspetti patogenetici della malattia si stanno evidenziando da alcuni studi che prendono in considerazione la disregolazione di alcune vie geniche che si presenta in soggetti con mutazioni puntiformi del gene SOD1 che codifica per la superossido dismutasi tipo 1,un enzima citosolico presente in tutte le cellule eucariote con importanti capacita’antiossidanti.
Si e’ visto che nei paziente con la forma mutante di SOD1 indipendentemente dall’attivita’ del enzima in questione all’interno del motoneurone si presenta una disregolazione di circa altri 21 geni che operano in alcune vie che possono rendere conto della degenerazione del motoneurone.
In primis si prende in considerazione la via biosintetica della D/L serina.
La serina e’ uno degli aminoacidi essenziali,la sua presenza e’ importante nella sintesi di purine e pirimidine,rappresenta un precursore di glicina e cisteina assumendo inoltre un ruolo importante nella biosintesi di sfingolipidi.La sua sintesi parte essenzialmente dal derivato glicolitico 3-fosfoglicerato nella reazione che segue:

La neutotossicita’ derivante da questo aminoacido e’ implicabile all’ induzione degli enzimi 3-fosfoglicerato deidrogenasi e fosfoserina fosfatasi comportando codi’ una disregolazione di questa via.I due isomeri cosi’ prodotti,l’ isomero L che funge da fattore trofico,sia all’isomero D(derivante dal’isomero L grazie all’enzima serina-racemasi) che risulta un coagonista dei recettori NMDA (recettori glutammato)determinerebbero un ruolo fondamentale nella patogenesi del danno.Il Il glutammato è il più importante neurotrasmettitore eccitatorio del S.N.C.. Il glutammato rilasciato dai neuroni presinaptici provoca la depolarizzazione dei neuroni postsinaptici principalmente mediante recettori NMDA (N-Metil-D-Aspartato)cosi’ Ca2+ e Na+si introducono nel neurone. L’up-regulation è considerata responsabile della neurotossicità del glutammato ed è dovuta ad un eccessivo afflusso di Ca2+ nella cellula, fenomeno che porta alla morte cellulare.

La seconda alterazione dimostrata riguarda l’attivazione della via classica del complemento che rispecchia un importante aspetto della neurodegenerazione rilevata anche in patologie importanti come Alzheimer e Parkinson.

Gli mRNAs per le tre subunita della proteina C1q(a,b,c) risultano fortemente indotti nei motoneuroni che esprimono la forma mutante di SOD1 e in aggiunta uno dei maggiori inibitori della cascata del complemento Daf1 risulta down regolato in questi pazienti.
Il Terzo ed ultimo aspetto patogenetico riguarda molecole coinvolte nella risposta danno/rigenerazione neuronale quali il CD44 e il fattore ATF3 che risultano indotte nei motoneuroni affetti.Il CD44 e’ una glicoproteina di membrana coinvolta nell’adesione e nella migrazione cellulare rappresentando il recettore per l’acido ialuronico potendo inoltre reagire con altri ligandi quali osteopontina,collagene e metalloproteasi.Per splicing si ottengono diverse isoforme con funzione diversa.
L’AFT3 e’ membro di una famiglia di fattori di trascrizione indotti in seguito allo stress tissutale.Esso regola la trascrizione legandi il DNA insieme alla proteina Jun.Si pensa che essi rappresentino un importante marker di danno e studi successivi valuteranno la possibilita’ di modulare queste molecole allo scopo di aumentare la risposta rigenerativa e rallentare cosi il decorso della malattia.
(-Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons
Christian S. Lobsiger, Séverine Boillée, and Don W. Clevelandh3)
The National Academy of Sciences of the USA

COMPLICATIONS

La sopravvivenza dei pazienti dipende dal mantenimento della funzione respiratoria (diaframma e muscoli intercostali) e dalla protezione delle vie aeree (riflesso della tosse e deglutizione), in quanto la causa più frequente di morte è rappresentata dalla paralisi respiratoria progressiva con broncopolmonite ab ingestis.Nell’insieme si considera una sopravvivenza non superiore ai 4 anni.

THERAPY