Reserpine pathways involved in lifespan extension, and hence the

Reserpine is a natural plant alkaloid purified from the roots of the plant Rauwolfia serpentina.  Ancient Indians had known and used it to treat snakebites and insanity (Lopez-Munoz et al., 2004).  Reserpine had also been tested as an antipsychotic and antihypertensive (Vakil, 1949; Bleuler and Stoll, 1955). Recently, this drug has been reported to extend lifespan and alleviate A? toxicity in C.elegans (Srivastava et al., 2008; Arya et al., 2009). Various pathways like reduced insulin signaling, calorie restriction and hormesis are established pathways of lifespan extension in worms (Olsen et al., 2006), flies (Hercus et al., 2003), mammalian cell culture and mice (Kenyon, 2005). However, reserpine did not utilize any of these known pathways to bring about lifespan extension in wild-type worms (Srivastava et al., 2008).

In the present study, wild-type worms (N2) when treated with 60uM reserpine increased the lifespan of worms by 26% (Fig 4.1.1) and also provided significant protection in the characteristic progressive paralysis with aging phenotype of AD model worms (Fig 4.1.2). This result was in concurrence with the study by Srivastava et al (2008) who had also reported an increase in worm’s lifespan on reserpine treatment. In addition to longevity, they also observed reserpine treated WT worms to have improved locomotion, pharyngeal pumping and thermotolerance. The study had reported that reserpine acts independent of the insulin-like signaling pathway and the calorie restriction pathway, the two most widely studied pathways involved in lifespan extension, and hence the mechanism of reserpine action needs to be established.   Earlier studies by Cohen et al (2006) and Morley et al (2002) had shown that a correlation exists between aging and neurodegenerative disease. In fact, reserpine reduces the plaque forming A?42 in the Tg2576 mouse model of AD and improved the working memory in the 5XFAD AD Tg mouse model (Go et al., 2013, Vasantharaja et al., 2016).

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It is known through several studies that lifespan and A? toxicity are dependent on the insulin signaling pathway. Thus extension of worms’ lifespan and protection against the paralytic effects of A? toxicity by reserpine, in the present investigation, indicated that the beneficial effects of reserpine are possible through a similar pathway.

A loss-of-function genetic mutant has reduced or no activity of the gene when mutated. An increased lifespan in these mutants with reserpine indicates that the absence of the gene has no significant effect on reserpine action.  On the other hand, when such mutants show reduced or no extension in lifespan, it implies that due to the absence of the gene, the drug is unable to bring about its effect and is important for drug’s action.

Impaired neurotransmission in organisms has been one of the many causes of aging and neurodegenerative diseases as evidenced in several studies. Impaired or excessive neurotransmission is implicated as a major contributor to NDs (Garcia et al., 2007; Pigino et al., 2003) and reduced cholinergic neurotransmission defect is one of the major hallmarks of AD pathology affecting functions such as cognition and behavior (Tabet, 2006; Liu et al., 2007). Additionally, serotonin receptors have also been linked to neurodegenerative disease and aging (Murakami and Murakami, 2007). These indicated neurotransmission as a promising mechanism through which reserpine could act.

unc-104 is a loss-of-function mutant for kinesin-1, which is responsible for the transport of synaptic vesicles along the axon. These mutants have a defective synaptic transmission as the synaptic vesicles accumulate in the neuronal cell bodies (Hall and Hedgecock, 1991). This abnormality results in a phenotypic expression as worms’ inability to move. Lack of RMLE in unc-104 (kinesin-1) mutants suggests that reserpine could have been taken up by the worms, but was unable to influence the longevity due to the impaired neurotransmission (Fig 4.1.1.1, Table 4.1.1). Thus neurotransmission is involved in RMLE.

Acetylcholine (ACh), the major excitatory neurotransmitter present in the neuromuscular junction (NMJ), carries out the motor functions in worms. Reserpine treated worms have been shown to have improved locomotion (Srivastava et al, 2008). Also, acetylcholine and acetylcholine receptors have been implicated in AD (Tabet, 2006). In order to identify the specific small molecule neurotransmitter in reserpine action, acetylcholine was first evaluated. cha-1 and unc-17, mutants for acetylcholine synthesis and transport, are hypomorphic mutants and are involved in presynaptic cholinergic neurotransmission. These mutants release very low amounts of ACh at the synapse (Rand and Russell, 1984). Both cha-1 and unc-17 worms are short-lived per se, with reserpine having no significant effect in enhancing their lifespan (Fig 4.1.1.2a). Reserpine treatment showed only 3% extension in the lifespan of cha-1 mutants, while a reduction of 35% in the mean lifespan in unc-17 worms was recorded (Table 4.1.1).

The short life of these worms could be due to the low ACh concentrations in their NMJ. Also, it has been reported that null mutants for ChAT and VAChT are lethal and non-viable (Alphonso et al, 1993); this indicates that ACh is essential for the viability of worms. It is known that acetylcholine synthesis decreases during the aging process (Gibson and Peterson, 1981) and modulation of acetylcholine levels also influence the dauer formation in worms (Lee et al., 2014). The reduced lifespan of these mutants with reserpine can also be considered as accelerated aging indicating that reserpine could be reducing the ACh concentrations further causing the worms to age and die faster and thus, acetylcholine is important for reserpine to bring about extension in lifespan in worms.

The role of neurotransmission and, specifically acetylcholine for reserpine mediated protection in A? toxicity was confirmed by aldicarb assay on the AD model worms, which is an established assay to study the defects in synaptic transmission, specifically, presynaptic cholinergic defects (Rand and Russell, 1985; Opperman and Chang, 1991; Mahoney et al, 2006; Nguyen et al, 1995). Aldicarb is an acetylcholinesterase inhibitor, which prevents the enzyme acetylcholinesterase to break down ACh at the synapse. In the presence of aldicarb, the ACh at the synapse starts accumulating which causes over-excitation of its receptors. This results in hypercontraction of muscles, leading to paralysis and even death of worms.

A mutant or worm with decreased ACh at synapse requires longer exposure to aldicarb to become paralyzed while the worms with enhanced ACh release will paralyze faster. Therefore the sensitivity of worms to aldicarb can deduce whether the worm has increased or decreased cholinergic signaling. As observed, aldicarb treated AD (CL2006) worms paralyzed earlier (Fig 4.1.2.1, Table 4.1.2), which indicates the accumulation of ACh at the synapse. Exposure of AD worms to reserpine, in presence of aldicarb, provided some resistance against aldicarb-induced paralysis. These findings further add to the evidence that reserpine reduces the concentration of ACh which prevents the worms from paralyzing. The lack of lifespan extension in cha-1 and unc-17 mutants, and the protection in AD worms against aldicarb-induced paralysis, implies that (i) reserpine modulates cholinergic neurotransmission, and (ii) this modulation occurs at presynaptic level.

To validate this and determine the effect of reserpine on the postsynaptic component, the nicotinic acetylcholine receptor (nAChR) mutants in worms were screened.  C.elegans body wall muscles express two types of ACh receptors. (i) Levamisole sensitive heteromeric receptors comprising ? and ? subunits. (ii) Homomeric receptors, made of only ? subunits, are levamisole insensitive but nicotine sensitive (Richmond and Jorgensen, 1999). unc-63 and acr-16 belong to two different classes of acetylcholine receptors expressed in worms and showed a significant reserpine mediated lifespan extension. Reserpine increased the average lifespan of unc-63 worms by 37% and that of acr-16 mutant by 40% (Fig 4.1.1.2b, Table 4.1.1), indicating that it acts independent of the acetylcholine receptors, in the absence of which reserpine can bring about extended lifespan. These receptor genes had no direct role in the enhancement of lifespan by reserpine.

Further, in order to determine the role of nAChRs in reserpine’s protective action against A? toxicity, reserpine’s effect in AD worms in the absence of unc-63 (AD;unc-63) was evaluated. unc-63 worms in itself showed progressive paralysis with age and reserpine could provide protection (~30%) against this (Fig 4.1.2.2b, Table 4.1.2). Reserpine also provided a delay in ~15% AD;unc-63 population despite the complications of unc-63 paralysis. Additionally, sensitivity of AD worms to levamisole in presence of reserpine (Fig 4.1.2.2a, Table 4.1.2) confirmed that reserpine acts independently of nAChRs.

In C.elegans, nAChRs are responsible for worm body muscle contraction on cholinergic stimulation.  One class of nAChRs is levamisole sensitive.  It is well known that levamisole is a more potent agonist than acetylcholine in nematode nAChRs (Lewis et al., 1980).  Levamisole activates nAChRs and leads to subsequent contraction of body wall muscles and paralysis.  Levamisole resistance indicates a postsynaptic cholinergic neurotransmission modulation. Both the aldicarb and the levamisole assay together, help to identify the location where the modulation is taking place. Worms with a defect in post synaptic components are resistant to levamisole, while those with pre synaptic mutations show similar rate of paralysis as their controls. Also if a worm is resistant to aldicarb but is sensitive to levamisole, then its presynaptic genes are targeted; but if a worm is resistant to both aldicarb and levamisole, it could be due to a mutation in its post synaptic gene (Mahoney et al, 2006).

In this study, (i) absence of RMLE in presynaptic cholinergic mutants cha-1 and unc-17 (Fig 4.1.1.2a), (ii) a significant lifespan extension in postsynaptic mutants acr-16 and unc-63 (Fig 4.1.1.2b) and (iii) resistance against aldicarb paralysis (Fig 4.1.2.1) while sensitivity towards levamisole in AD worms (Fig 4.1.2.2a) strengthened that reserpine acts presynaptically and independent of nAChRs. Also, it provides extension to lifespan and protection against A? toxicity, both by modulating the levels of ACh.