Properties of mAbs raised against A1NTX.
In the present review, we describe here experimental comparative and beneficial effects of botulinum neurotoxin A (ANTX) between subtypes A1 and A2 in the pathology of movement disorders, particularly rat Parkinson’s disease model. We and other laboratories have shown the beneficial effects, and this novel strategy for intractable brain disorders might confer potent and safety therapy in bedside. First, we show the characteristics of ANTXs in the genetic aspects of these subtypes, and our intriguing findings of immunological profiles in the subtypes between A1NTX and A2NTX. Then, we state the distinct diffusion in the body between A1NTX and A2NTX. Importantly, we describe that the intra-brain treatment of small animals with A2NTX subtype results in improvements of pathologies more effectively and provides greater safety than those of A1NTX in a rat 6-OHDA Parkinson’s disease (PD) model. Finally, we represent that the different efficacies between ANTXs are likely due to each localization in the brain; A2NTX is strictly limited in the injected regions, while A1NTX diffused other brain regions. Thus, therapeutic avenue using A2NTX in incurable PD including other movement disorders could be a druggable target in the future.
- botulinum neurotoxin type A
- Parkinson’s disease
NTXs are released from
Due to their high efficacy, tolerance, longevity, and safety property, botulinum toxins are the most widely used therapeutic proteins. Most of the toxins have several subtypes based on amino acid sequence variability. The type A toxins have been subclassified into 10 subtypes (A1–A10) . Especially, the toxin products used as treatment for neurologic disorders are LL toxin and NTX, produced by botulinum toxin subtype A1. The other subtype of toxins is not used clinically, however has been conducted in researches. We have been studying the biological characteristic and pharmacology of A2 toxin.
Several species of botulinum neurotoxin are known to act on cholinergic terminals of the peripheral neuromuscular junction and the central nervous system (CNS) [3, 4, 5]. NTXs cause robust inhibition of the voluntary nervous circuits by blocking the release of acetylcholine (ACh) . The therapeutic application of A1NTX for neurological disorders such as bradykinesia, urinary dysfunction, hemifacial spasm, and cervical dystonia is well established . The type A organisms have been classified into 10 subtypes (A1 to A10) based on the amino acid sequence variability of NTX . All 10 subtypes bind to presynaptic protein SNAP-25 with similar affinity, but A1NTX and A2NTX cleave SNAP-25 more efficiently than that of other subtypes [4, 8, 9].
Recent studies investigated the direct administration of ANTX to the CNS as a therapeutic strategy for the treatment of neurological disorders [3, 4, 5]. Parkinson’s disease (PD) is characterized by imbalanced cholinergic hyperactivity in the striatum of affected individuals [10, 11]. Interruption of ACh release in the striatum by direct injection of BoNT/A has been reported in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD .
This paper will review the recent advance in the genetic, immunological, diffusion in the body and experimental animal model of PD in botulinum toxin A.
2. Genetic diversity between A1NTX and A2NTX
2.1. Genetic diversity of gene clusters encoding ANTX complexes
The neurotoxin and nontoxic protein genes are defined as the NTX gene cluster. There are two types of nontoxic components of gene organization (HA and Orfx clusters), and
2.2. Immunological differences between A1NTX and A2NTX
The difference in amino acid sequence between subtype A1 and A2 toxins’ light chain is 5%, while the difference in heavy chain is 13%. The similarity of heavy chain is lower than light chain. These differences appear to indicate that characteristic antigenicity in the heavy chain is more conserved than that in the light chain . Differences in antigenicity among subtypes were evaluated using monoclonal and polyclonal antibodies [20, 21, 22, 23]. Among eight and seven monoclonal antibodies against A1NTX and A2NTX, respectively, each of which recognized different epitopes, each three specifically reacted with A1NTX and A2NTX. Neutralizing single monoclonal antibodies against A1NTX and A2NTX that recognized LC, HN, or HC have been reported, respectively (Tables 1 and 2). Each neutralizing antibody mostly neutralized only toxins of their own subtypes. It is suggested that the epitopes of neutralizing are present in every domain of both subtypes. The 3B10 and 5G2 that are reacting with LC and HN, respectively, specifically recognized and neutralized A2NTX. These monoclonal antibodies recognizing epitopes are considered to function as A2NTX properties. In type B, differences in biological activities among the subtypes B1, B2, and B6NTX appeared to be attributable not only to the function in HC but also to the function in HN . For binding of monoclonal antibodies to NTX, KD values of 1F11 for A1NTX were 500 hold higher than that for A2NTX and only neutralized A1NTX. However, the KD values of 5C7 for A2NTX were 16 hold higher than that for A1NTX did not neutralize both NTXs. The neutralization of monoclonal antibody did not correspond to its affinity. And, OD values obtained by ELISA did not necessarily correlate with KD values (Table 3).
Type A antitoxin in standard and therapeutic preparation is a polyclonal antibody purified from immunized sera with A1NTX; however, there was no report on the reactivity of the standard type A antitoxin with other subtype toxins. The A1 antitoxin had equivalent potency both the A1NTX and A2NTX; however, neutralization titer of A2 antitoxin was 4–9 hold higher against A2NTX than against A1NTX. It seems that the difference between the antibody titers against the test NTX was due to the standard antitoxin having different reactivities with the NTXs. The binding analysis comparing these antitoxins and NTXs by SPR showed that the A1 antitoxin had a higher binding affinity and slower dissociation speed with the A1NTX than with the A2NTX. The A2 antitoxin showed a higher binding affinity than with the A1NTX . Although these NTXs show a low level of sequence difference, they have marked a difference in antigenicity, and antitoxin preparation should be used for each subtype’s diagnosis and therapy of botulism.
3. Diffusion into the body of botulinum toxins A1 and A2
Botulinum toxins type A have been researched and developed for use as important therapeutic agents for neurological disorders such as blepharospasm, hemifacial spasm, various dystonias, and overactive bladder [7, 25]. Botulinum toxin type A products, which are used as treatment for neurologic disorder, are produced from LL toxin or NTX derived from subtype A1 organisms . The toxins show high-level efficacy at very low doses, but their adverse effects are becoming an issue. In the treatment for torticollis, cervical dystonia, and cosmetic cases, patients showed dysphagia or respiratory compromise [27, 28, 29]. In clinical studies of treatment for spasm, patients who received high-dose toxin showed weakness around the site of administration as well as symptoms of botulism [30, 31, 32]. The A1 toxins spread to distant regions is considered to be due to transport via the body fluid or nerves [33, 34, 35]. In addition, A1 toxin was reported to transport via a retrograde axonal route in visual nerve and facial motoneurons in rats .
The first report of the diffusion of A2 toxin in the body was grip strength study in mice to compare with A1 toxin . This study was evaluated by measurement of contralateral grip strength as indicator of toxins’ diffusion. The toxins used were A1 L + LL toxin, onabotulinumtoxinA (A1LL toxin), and A2M toxin and were injected into one side of the gastrocnemius muscle, and grip strength of the contralateral hind leg was measured. The study evaluated that the doses causing a 20% reduction in the grip strength before injection were calculated and these values were termed the 20% toxic doses (TD20). The TD20 of A1L + LL toxin, A1LL toxin, and A2M toxin were 17.0, 16.2, and 37.3 U/kg, respectively. The grip strength test was conducted for change in toxins’ forms, measurement sites, and animal species . The grip strength test using rats’ forelegs was conducted using A1 neurotoxin (A1NTX), A1LL toxin, and A2NTX (Figure 1). The study evaluated that 50% toxic doses (TD50), which caused a 50% reduction in the grip strength before injection, were calculated. The TD50 values of A1NTX, A1LL toxin, and A2NTX were 7.54, 6.35, and 15.62 U/head, respectively. These results indicated that A2NTX required higher dosage than A1 toxins to relax on the contralateral muscle and suggested that A1 and A2 toxins have different diffusions in the body.
Why do these toxins make a difference in diffusion in the body? The pathway of A1 and A2 toxins was physiologically investigated in the immunohistological study .
Spinal cords (bilateral ventral and dorsal horns), in which A1NTX and A2NTX were injected into the gastrocnemius muscle, were strained using botulinum toxin type A-cleaved SNAP-25 (cSNAP-25). The L5 nerve dominantly innervates the gastrocnemius muscle. The A1NTX was observed to have a strong immunoreactivity for cSNAP-25 in the ventral and dorsal horns of the spinal cord not only at the segmental level of L5 ipsilateral to the peripheral toxin injection site but also to a lesser extent on the contralateral side (Figure 2A). The A2NTX was observed to have a strong immunoreactivity at the L5 spinal segment ipsilateral side as A1NTX but to a lesser extent on the contralateral side than A1NTX (Figure 2B). In addition, the ventral horns stained for cSNAP-25 at the L5 spinal segment in the toxin-treated rats were compared by optical density measurements. In both the ipsilateral and contralateral ventral horns, cSNAP-25 labeling in rats injected with A1NTX was spread wider than with A2NTX (Figure 2C).
The diffusion of A2NTX in the body summarized the previous reports as follows (Figure 3). After unilateral intramuscular toxin injection, the catalytically active toxin can be axonally transported to the spinal cord through motor and sensory nerves. Subsequently, the toxin can spread throughout the gray matter of the spinal cord, including the bilateral ventral and dorsal horns, via a transcytosis (cell-to-cell trafficking) mechanism by which a ligand penetrates the neuron at one side, followed by its movement and release at the opposite end, with possible uptake by second-order neurons. Differential delivery routes by which injected A1NTX and A2NTX affect contralateral muscles have also been proposed as A1NTX is transported almost equally to the contralateral muscles via this neural pathway and the blood circulation, while A2NTX is mainly transported to contralateral muscles via the bloodstream only at higher doses.
4. Therapeutic application of botulinum toxins A1 and A2 in Parkinson’s disease
Parkinson’s disease (PD) is one of the most common movement disorders and is characterized by a progressive degeneration of nigrostriatal dopaminergic signaling, which leads to the unbalanced release of acetylcholine in the striatum . The disturbance of these neuronal circuits elicits parkinsonian motor symptoms with muscular dysfunctions, such as resting tremor, spontaneous dystonia, akinesia, sialorrhea, urinary dysfunction, and pain [10, 39]. While palliative therapies for PD subjects having sialorrler and urinary dysfunction using onabobotulinamtosinA (nealy equal to A1NTX) are going in bedside , there is currently a lack of curative therapies using ANTXs.
Several studies demonstrated that the intrastriatal injection of A1NTX reduces pathologic behavior in the rat 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease model (rat 6-OHDA PD model) [11, 40]. These studies demonstrate the feasibility of clinical A1NTX application to treat PD without adverse side effects such as memory dysfunction [11, 40]. However, it is not clear which A1NTX has the greatest efficacy for treatment of PD. Therefore, we first compared the effect of A1NTX with that of A2NTX on pathogenic rotation behavior and in vivo cleavage of striatal SNAP-25 in the 6-OHDA PD rat model.
As a result, intrastriatal treatment of 6-OHDA-lesioned rats with A1NTX or A2NTX significantly reduced the pathogenic rotation behavior in a dose-dependent manner (Figure 4). The highest tested dose of A1NTX (1 ng) conferred significant reduction of pathogenic behavior, as did all tested A2NTX doses (0.1, 0.5, and 1 ng). These results suggest that A2NTX has more potent inhibition of ACh release in the striatum than that of A1NTX . Indeed, intrastriatal injection of the 6-OHDA-lesioned rats with A1NTX or A2NTX caused a dose-dependent decrease in the level of full-length SNAP-25 in the striatum . These results support the observed effects of A1NTX and A2NTX on rotation behavior (Figure 4). Additionally, we investigated the localization of cleaved SNAP-25 and choline acetyltransferase in the ANTX-treated striatum by performing fluorescent immunocytochemical analysis . These results indicate that A2NTX has greater efficacy for SNAP-25 cleavage in striatal terminals than that of A1NTX. Therefore, their dose-dependent efficacies in the striatum appear to differ, although the therapeutic effects of both toxin species on reducing pathologic rotation behavior in a PD rat model are likely due to their cleavage of SNAP-25 .
Several side effects have been reported after therapeutic treatment with ANTXs for cervical dystonia and cosmetic cases, such as dysphagia and respiratory compromise [28, 29]. Our studies also demonstrated that the effects of botulinum toxin could spread from the injection site to other areas of the body causing symptoms similar to those of botulism ; A1NTX, but not A2NTX, was transported via axons to the contralateral side after injection into the foreleg muscles as described in Section 3. These results suggest that A2NTX may have a wider safety margin than that of A1NTX for therapeutic applications for PD. Thus, we investigated side effects after intrastriatal injection of either A1NTX or A2NTX in the rat 6-OHDA PD model .
To investigate the distribution of A1NTX or A2NTX in the striatum, an immunofluorescent analysis of the cleaved SNAP-25, which is produced by ANTXs, is performed. The area of survey is shown in Figure 5A. Compared to the treatment with vehicle control (Figure 5B), the treatment with A1NTX increased the cleaved SNAP-25 in both the ipsilateral and contralateral striata (Figure 5C and E). In contrast, for A2NTX, the cleaved SNAP-25 signals were observed only in the ipsilateral striatum (Figure 5D and F). These results indicated that A2NTX was retained at the injection site, whereas A1NTX was diffused into the contralateral striatum.
Indeed, the previous study showed that ANTXs were retrogradely transported by central neurons and motoneurons and were then transcytosed to afferent synapses. The SNAP-25 cleaved by ANTXs was observed in the contralateral hemisphere after unilateral ANTX injection to the hippocampus [12, 43]. Moreover, this finding is supported by our findings showing that A1NTX injected into the foreleg muscles was transported via axons to the contralateral side more readily than A2NTX as indicated in Section 3.
Furthermore, we evaluated changes in body weight as an index of the adverse effects of ANTX application. Body weights were measured 1 and 9 days after the 1.0 ng ANTX injection. Treatment with A1NTX resulted in significant loss of body weight compared to both the vehicle and A2NTX groups (Figure 6). Together with Figures 3 and 5, these results suggest the possibility that A1NTX, but not A2NTX, diffuses into the contralateral hemisphere leading to dysfunction in food/water intake.
Why does the difference between A1NTX and A2NTX arise in a rat PD model? Interestingly, A2NTX enters neuronal cells faster than A1NTX . Additionally, we found that A1NTX and A2NTX have distinctly different distributions in the peripheral neuromuscular system in Section 3. Unfortunately, these findings only are not sufficient to explain the differences of ANTX subtypes in vivo. Thus, further studies are needed to elucidate the variation among ANTXs from the views of genetic, immunological, and neurological aspects.
Considering the available evidence, it can be concluded that (1) the isolates associated with infant botulism were epidemiologically divided into NTXA gene cluster types. And, A1NTX and A2NTX have marked a difference in antigenicity. (2) A2NTX caused less muscle flaccidity of nontoxin-treated muscle than A1 toxins. The variation in the amino acid sequence between A1NTX and A2NTX causes the difference in the spreading pathways. (3) A2NTX provides anti-PD effectiveness more effectively and confers greater safety than those of A1NTX. These findings might open a new therapeutic avenue for not only PD subjects but be useful also for application to other parkinonisms.
We entirely thank Drs. Shunji Kozaki and Ryoji Kaji (Tokushima University) for both their excellent discussion and encouragement to undergo this review. These works were supported in part by a grant-in-aid (C; 22580339, C; 25450428, and B; 16H05029, to H.N.) and (B; 21380188, S.K.) from the Japan Society for the Promotion of Science.
Conflict of interest
The authors declare no conflict of interest.
|ANTX||Clostridium botulinum neurotoxin subtype A|
|CNS||central nervous system|
|A1NTX||ANTX subtype A1|
|A2NTX||ANTX subtype A2|
|GPS||gelatin phosphate buffer (pH 6.2)|
|KD values||the affinity constant calculated as dissociation (kd) rate constant/association (ka) rate constant|
|LD50||50% lethal dose|
|Orfx||unknown function open reading frame gene|
|SNAP-25||synaptosomal-associated protein of 25 kDa|