**3.4 Rural–urban ratio of crime rate**

A total of 39 (37.1%) cases of crimes against the elderly women have been reported in rural area as compared to 64 (62.9%) cases of crimes reported in urban area. The crime rate of an area decides level of fear of crime. The crime rate of an area reveals anxiety about crime and it is also found that crime rate is higher in urban areas and urban people are more fearful than rural areas [16] (**Figure 2**).


**Author details**

Oğuzhan Kahraman, Zekeriya Safa İnanç\*, Huzur Derya Arık and Mustafa Selçuk Alataş Faculty of Veterinary Medicine, Department of Animal Nutrition and Nutritional Diseases, Selcuk University, Konya, Turkey

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Unilateral Botulinum Neurotoxin-A Injection into the Striatum of C57BL/6 Mice Leads to a Different Motor Behavior Compared with Rats**

#### **Veronica Antipova 1,2, Andreas Wree 1,\*, Carsten Holzmann 3, Teresa Mann 1, Nicola Palomero-Gallagher 4,5, Karl Zilles 4,5,6, Oliver Schmitt 1 and Alexander Hawlitschka 1**

Received: 5 July 2018; Accepted: 15 July 2018; Published: 17 July 2018

**Abstract:** Different morphological changes in the caudate-putamen (CPu) of naïve rats and mice were observed after intrastriatal botulinum neurotoxin-A (BoNT-A) injection. For this purpose we here studied various motor behaviors in mice (*n* = 46) longitudinally up to 9 months after intrastriatal BoNT-A administration as previously reported for rats, and compared both outcomes. Apomorphine- and amphetamine-induced rotational behavior, spontaneous motor behavior, as well as lateralized neglect were studied in mice after the injection of single doses of BoNT-A into the right CPu, comparing them with sham-injected animals. Unilateral intrastriatal injection of BoNT-A in mice induced significantly increased contralateral apomorphine-induced rotations for 1 to 3 months, as well as significantly increased contralateral amphetamine-induced rotations 1 to 9 months after injection. In rats (*n* = 28), unilateral BoNT-A injection also induced significantly increased contralateral apomorphine-induced rotations 3 months after injection, but did not provoke amphetamine-induced rotations at all. Lateralized sensorimotor integration, forelimb preference, and forelimb stepping were significantly impaired on the left side. The differences in motor behaviors between rats and mice may be caused by different BoNT-A effects on cholinergic and catecholaminergic fibers in rat and mouse striata, interspecies differences in striatal receptor densities, and different connectomes of the basal ganglia.

**Keywords:** botulinum neurotoxin-A; basal ganglia; interspecies differences in motor behavior; mouse; rat; interneurons

**Key Contribution:** We investigated the effect of intrastriatal BoNT-A application on motor behavior in naïve mice for the first time. To interpret the differences of the BoNT-A effect in mice and rats, multi-receptor fingerprints of Wistar rats and C57Bl/6 mice as well as their basal ganglia connectomes were compared.

#### **1. Introduction**

Parkinson's disease (PD) is a common chronic progressive age-related neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and, subsequently, of dopamine in the caudate-putamen (CPu) [1–3]. Dopamine deficit causes a profound impairment of neuronal circuits in the basal ganglia [4–7], and particularly an increased release of acetylcholine by tonically active striatal interneurons [8–12]. Anticholinergic drugs are used to antagonize striatal hypercholinism in PD, but this treatment often elicits adverse side effects [13–15]. In order to circumvent general anticholinergic drug effects, we tested a local intrastriatal injection of botulinum neurotoxin-A (BoNT-A) [16–20].

Indeed, in the experimental 6-OHDA-induced hemiparkinsonian (hemi-PD) rat model, intrastriatal application of BoNT-A abolished apomorphine-induced rotational behavior—most probably by blocking acetylcholine, the release of cholinergic terminals, and/or inducing changes in receptor densities [16–23]. Moreover, the unilateral intrastriatal BoNT-A injection induced rotational behavior in naïve rats without 6-OHDA-induced hemi-PD. Following the injection of 1 ng BoNT-A into the right CPu, rats showed 2–4 apomorphine-induced rotations per min in the direction of the injection site for two months, and rotated tentatively to the contralateral side thereafter [19].

To date, behavioral effects of intrastriatal BoNT-A injections were studied in rats, but not in mice. This would be interesting, as comparative morphological studies of the striata of BoNT-A-injected rats and mice striata showed obvious differences in the structural changes of choline acetyltransferase-immunoreactive (ChAT-ir) and tyrosine hydroxylase-immunoreactive (TH-ir) fibers [19,20,24], in addition to similarities concerning unchanged CPu volume and the number of cholinergic interneurons. Rats and mice differed in the induction of BoNT-A-induced varicosities: both kinds of these varicosities were found in rats [16,17,19–21], whereas only ChAT-ir BoNT-A-induced varicosities were observed in mice [24].

As naïve mice and rats reacted morphologically in a different manner following intrastriatal BoNT-A injection, we sought to determine whether these species differences would also hold true for motor behavior. Thus, mouse motor behavior was scored after unilateral BoNT-A injection into the right CPu using apomorphine- and amphetamine-induced rotations. Spontaneous behavior was tested using the cylinder and stepping tests, lateralized sensorimotor activity by the corridor task, and cerebellar ataxia by hindlimb clasping. To evaluate temporally restricted effects, mice were tested up to 9 months after BoNT-A injection. A potential dose-dependency was evaluated by using dosages of 25 and 50 pg BoNT-A.

It is known that mice are very sensitive to botulinum toxins and also differ in many respects from rats [25–33]. However, there are currently no studies evaluating the behavioral outcome of intrastriatally applied BoNT-A in naïve mice. Moreover, the effect of BoNT-A in naïve mice must be understood prior to evaluation of intrastriatal BoNT-A application in mice models of hemi-PD and genetic PD. The BoNT-A dosage used for intrastriatal injection for the treatment of experimental hemi-PD in rats was based on the LD50 and recent intracerebral BoNT-A injections [34–38]. In Wistar rats we found a well-tolerated and effective dosage at 1 ng BoNT-A per CPu, whereas 5 ng were mortal [19]. In the C57BL/6 mice, dosages of 25–50 pg BoNT-A per striatum were found to be appropriate [24].

Studying the behavioral effect of unilateral intrastriatal BoNT-A application in naïve mice also seemed to be important, since the effect of therapeutic BoNT-A applications is explored in increasingly studied genetic Parkinsonian mouse models [39–45].

#### **2. Results**

#### *2.1. Body Weight*

Changes in body weight were evaluated as an index of general adverse effects of BoNT-A application. The mice weighed 18–24 g at the time of BoNT-A or vehicle injection. Thereafter, body weight increased in all experimental groups (Figure 1). Increase in body weight over time was significantly (F2, 50 = 15.5999, *p* < 0.001) lower in mice injected with BoNT-A than in those receiving vehicle substance. However, 9 months after BoNT-A injection, the 25 pg BoNT-A mice weighed 31.85 ± 0.37 g (mean ± SEM), the 50 pg BoNT-A mice 33.72 ± 0.40 g, and thus did not differ from the sham group, which weighed 34.66 ± 0.35 g (Figure 1).

**Figure 1.** Body weight over time. Asterisks indicate significant differences (25 pg, *n* = 15; 50 pg, *n* = 20) compared to the sham group (*n* = 11) (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). Data are means ± SEM.

#### *2.2. Apomorphine-Induced Rotations*

#### 2.2.1. Mice

In order to test the effect of a unilateral intrastriatal BoNT-A injection on drug-induced motor behavior we used apomorphine- and amphetamine-induced rotation tests. Half a month after injection of 25 or 50 pg BoNT-A or vehicle into the right CPu mice showed no apomorphine-induced rotation behavior (Figure 2A). Vehicle application did not induce rotational behavior in the subsequent 9 months. However, both applications of 25 and 50 pg BoNT-A resulted in significant (F2, 50 = 6.150, *p* = 0.004) anti-clockwise, i.e., contralateral, apomorphine-induced rotations with net rotations of about 2 per minute after a post-injection survival of 1 and 3 months (Figure 2A). Six and 9 months after BoNT-A, rotational behavior decreased to values similar to those observed in the vehicle-treated group.

**Figure 2.** (**A**) Apomorphine- and (**B**) amphetamine-induced rotations in mice treated with intrastriatal BoNT-A (25 pg, *n* = 15; 50 pg, *n* = 20) or vehicle (*n* = 11). (**A**) BoNT-A in either dosage caused a significant increase of the apomorphine-induced turning rate 1 and 3 months after injection. (**B**) BoNT-A in both dosages resulted in significantly increased amphetamine-induced rotations 1–9 months after injection, while sham injections did not change rotational behavior. Asterisks indicate significant differences compared with the sham group (\*\* *p* < 0.01, \*\*\* *p* < 0.001). Data are means ± SEM.

#### 2.2.2. Rats

Following apomorphine injection rats significantly rotated contralaterally to the intrastriatal BoNT-A application with rotations of approximately 1.5 to 2 per minute 3 months after 1 ng BoNT-A (Figure 3A).

**Figure 3.** (**A**) Apomorphine- and (**B**) amphetamine-induced rotations in rats intrastriatally injected with BoNT-A (*n* = 22) or vehicle (*n* = 6). (**A**) 1 ng BoNT-A caused a significant anti-clockwise apomorphine-induced turning 3 months after injection, (**B**) but did not influence amphetamine-induced rotation behavior significantly 1–9 months after BoNT-A as compared to the sham group. Asterisks indicate significant differences compared with the sham group (\*\* *p* < 0.01). Data are means ± SEM.

#### *2.3. Amphetamine-Induced Rotations*

#### 2.3.1. Mice

After intrastriatal injection of 25 or 50 pg BoNT-A, the mice showed significantly increased (F2, 48 = 15.360, *p* < 0.001) amphetamine-induced rotations compared to the vehicle group from 1 to 9 months post injection (Figure 2B). Whereas application of BoNT-A was associated with strong amphetamine-induced anti-clockwise net rotations of approximately 4–10 per min, vehicle injection resulted in clockwise rotations of approximately 3 per min (Figure 2B).

#### 2.3.2. Rats

In rats, amphetamine application revealed no significant effect of unilateral intrastriatal BoNT-A as compared with vehicle injection (Figure 3B).

#### *2.4. Cylinder Test*

The cylinder test evaluates asymmetries in spontaneous forelimb use during exploratory activity in a novel environment. Sham injection did not induce any laterality of forepaw usage during the whole testing period up to 9 months; left and right forepaws were used equally often in the sham group (Figure 4A). In contrast, BoNT-A-injected mice, irrespective of the dosage and survival time, exhibited a significantly reduced (F2, 52 = 31.971, *p* < 0.001) use of the left forepaw of about 40% (Figure 4A).

**Figure 4.** (**A**) The cylinder test of BoNT-A-injected mice (25 pg, *n* = 15; 50 pg, *n* = 20) of both dosages revealed a significantly lower use of the left forepaw at all time points compared with sham-injected mice (*n* = 11). (**B**) In the corridor task, BoNT-A-injected mice of both dosages (25 pg, *n* = 11; 50 pg, *n* = 12) retrieved pellets significantly less often from the left side compared to the sham group (*n* = 10). (**C**,**D**) The stepping test revealed constant adjusting steps of sham-treated mice (*n* = 10). (**C**) Right side BoNT-A-injected mice of both dosages (25 pg, *n* = 11; 50 pg, *n* = 10) displayed significantly decreased left forepaw adjusting steps 1–9 months after surgery, whereas (**D**) the number of adjusting steps of the right forepaw only differed significantly from the sham group one month after BoNT-A injection. However, the 50 pg BoNT-A group also showed significantly fewer right forepaw adjusting steps 9 months after injection. Asterisks indicate significant differences compared with the sham group (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). Data are means ± SEM.

#### *2.5. Corridor Task*

Lateralized sensorimotor integration was assessed using the corridor test, which depends on the rodent's ability to retrieve food from either side of its body. Animals of the vehicle group made an equivalent number of retrievals (about 50% of total retrievals from both the left and right sides of the corridor) over the whole testing period (Figure 4B). However, mice of both BoNT-A groups showed a significant neglect (F2, 40 = 11.506, *p* < 0.001) of the corridor side contralateral to the BoNT-A injection side. These mice retrieved only about 35% of the sugar pellets from the left corridor side during the whole observation period (Figure 4B).

#### *2.6. Stepping Test*

The motor activity of the forelimbs was estimated using a mouse-friendly version of the stepping test. Adjusting forepaw steps were measured on the injected (right) and noninjected (left) sides. Mice of the sham group made approximately 10–11 steps with either forepaw during the whole testing time (Figure 4C,D). Mice of both BoNT-A groups exhibited significantly fewer adjusting steps (F2, 42 = 99.773, *p* < 0.001) with their left forepaws up to 9 months after BoNT-A (Figure 4C). A reduction in adjusting steps with the right forepaw was seen in BoNT-A-injected mice of both dosages 1 month after BoNT-A (Figure 4D).

#### *2.7. Hindlimb Clasping*

We evaluated neurological abnormalities using the hindlimb clasping test. Mice of the two BoNT-A groups and the sham group did not show any pathological hindlimb clasping during the whole testing period from 1 to 9 months; the hindlimbs of all mice were consistently splayed outward and away from the abdomen resulting in an assigned score of 0.

#### **3. Discussion**

Previous studies of unilateral injections of BoNT-A into the CPu of male C57BL/6 mice showed that the number of ChAT-ir interneurons was unaltered in the CPu, and ChAT-ir BoNT-A-induced varicosities were visible. Both results are comparable with findings in the rat [16–20]. However, in contrast to rats, there were no TH-ir BoNT-A-induced varicosities in BoNT-A-injected mouse. As BoNT-A application seemed to have a different influence on cholinergic and dopaminergic axons in the mouse striatum [24], the CPu-associated motor behavior was studied. Moreover, a possible temporally occurring BoNT-A effect as seen in previous studies in rats [16–21] was evaluated by testing mice up to 9 months after BoNT-A injection. Dose dependency was studied by using dosages of 25 and 50 pg BoNT-A. As a significant dose dependency within the evaluated parameters was not obvious, the results of mice receiving dosages of 25 and 50 pg BoNT-A will be discussed together.

We found that unilateral intrastriatal BoNT-A injection in naïve mice led to significant contralateral amphetamine- and apomorphine-induced rotations as well as to significant impairments of the left (i.e., contralateral) side with respect to lateralized sensorimotor integration, forelimb usage, and forelimb stepping.

#### *3.1. Basal Ganglia Circuitry after BoNT-A Injection*

Locally injected BoNT-A is thought to act in two main ways in the striatum, where it blocks the acetylcholine release of the tonically active cholinergic interneurons [46–48], counteracts the D2 receptor upregulation found in hemi-PD rats, and reduces D2 receptor concentrations in naïve rats [22,23]. The assumed BoNT-A-induced reduction of acetylcholine concentration in the injected CPu reduces the firing activity of medium spiny neurons projecting to (i) the external globus pallidus, i.e., those which are part of the indirect basal ganglia loop, and (ii) to the internal globus pallidus, i.e., those which are part of the direct basal ganglia loop [49–53].

The effects of BoNT-A on the reduction of cholinergic transmission and of D2 receptor concentrations seemingly underlie movement initiation deficits, akinesia and reduced spontaneous use of the contralateral forelimbs [54–57].

#### *3.2. Body Weight*

Body weight increased in all three experimental groups from two weeks up to 9 months after intrastriatal BoNT-A or vehicle injection. However, mice injected with either of the BoNT-A dosages weighed less compared to the sham group two weeks after injection onwards, although the differences diminished after 9 months. These differences might point at a mild temporal toxicity of BoNT-A after the stereotactic injection.

#### *3.3. Spontaneous Motor Tests*

#### 3.3.1. Spontaneous Forelimb Use

The cylinder test evaluates locomotor asymmetry and forelimb use in rodent models of central nervous system disorders by assessing the innate drive to explore a novel environment by rearing and leaning their forepaw against the wall of the glass cylinder [58]. Unilaterally sham-injected mice used the left and right paws symmetrically, i.e., about 50% each. Right side intrastriatal application of BoNT-A significantly reduced the use of the left paw.

Seemingly, the impairment of motor initiation deficit for voluntary movements of the contralateral forelimb is a specific result of BoNT-A injection into the CPu since it decreases firing of GABAergic medium spiny neurons to the internal globus pallidus in the direct loop, and therefore inhibits the ventrolateral thalamic nucleus. On the other hand D2 receptor-bearing medium spiny neurons increase inhibition of the external globus pallidus in the indirect loop, and resulted in a more actively firing internal globus pallidus via reduced inhibition of the spontaneously active subthalamic nucleus. Therefore, the inhibited neurons of the ventrolateral thalamic nucleus are not able to sufficiently activate the premotor cortex via both loops.

#### 3.3.2. Sensorimotor Integration

The corridor task, originally designed to study unilateral sensorimotor integration impairments in rats [59,60], was adapted for experiments in mice [61]. Mice injected with 25 or 50 pg BoNT-A into the right striatum retrieved pellets significantly less often from the left side during the testing period up to 9 months after BoNT-A, whereas sham-injected mice behaved symmetrically. As is the case for the forepaw preference studied in the cylinder test, the motor initiation deficit for voluntary movements of the contralateral forelimb is probably a specific result of the BoNT-A injection into the CPu. Both the reduction of striatal cholinergic transmission and the reduction of D2 receptor density causes changes in the basal ganglia circuitry resulting in a reduced initiation of movements of the contralateral body via crossed motor efferents [57,62,63].

#### 3.3.3. Forelimb Adjusting Steps

In our study, we used the mouse-friendly version of the stepping test described by Blume et al. [64] and modified by Heuer et al. [65]. Right sided intrastriatal application of 25 and 50 pg BoNT-A clearly reduced the stepping frequency of the left forepaw up to 9 months after injection compared to sham group. A reduced initiation of movements of the paw contralateral to the BoNT-A application via crossed motor efferents is reasonable for the outcome of this behavioral test [66–68]. Interestingly, the intrastriatal BoNT-A injection into the right CPu also results in a short-term reduction of right paw adjusting steps. One month after vehicle injection mice made 10.8 ± 0.2 steps; at the same time the 25 pg BoNT-A group made 9.2 ± 0.2, and the 50 pg BoNT-A group 8.9 ± 0.2 steps. This phenomenon is not fully understood, as the right forepaw steps should be unaltered. The mouse-friendly design of the stepping test has also been applied in the mouse MTPT model [64] resulting in bilateral impairment, and also following different right side models of dopaminergic lesioning to induce unilateral parkinsonian-like symptoms [65,69,70]. Unilateral dopaminergic depletion, however, led to contradicting results in the same test. Heuer et al. [65] stated that the number of steps is reduced in both paws to about 80% compared to sham-injected mice irrespective of the experimental approach inducing unilateral or bilateral striatal dopamine reduction. In contrast, Glajch et al. [69] reported a ratio of contralateral-to-ipsilateral steps of about 0.2 after unilateral 6-OHDA lesion of the medial forebrain bundle. Boix et al. [70] reported a ratio of approximately 6 to 27%, depending on the 6-OHDA dosage used. Thus, both groups show a clear unilateral effect. However, neither Glajch et al. [69] nor Boix et al. [70] mention the absolute number of steps made by the ipsilateral paw, which would be important for comparison with our results.

#### 3.3.4. Hindlimb Clasping

Hindlimb clasping has been shown to occur in various neurodegenerative mouse models [71,72] including PD models [73,74]. All our mice, irrespective of BoNT-A or vehicle injection, never showed pathological hindlimb clasping during the whole testing period from 1 to 9 months. We interpret the absence of this pathological behavior in our model as indirect evidence that the BoNT-A dosages used are not generally toxic in the striatum.

#### *3.4. Drug-Induced Rotation Tests*

#### 3.4.1. Apomorphine-Induced Rotations

There is an apomorphine-induced rotational behavior of BoNT-A-treated mice and rats. Mice with 25 and 50 pg BoNT-A injected intrastriatally showed significant contralateral apomorphine-induced rotations after a survival of 1 and 3 months with rotations of about 2 per minute. Thereafter, rotational behavior decreased to values not significantly different from those of the vehicle group. These measurements corroborate data obtained in rats (Figure 3A), which also showed a significantly altered behavior with rotations of approximately 1.5 to 2 per minute 3 months after intrastriatal injection of 1 ng BoNT-A.

Apomorphine-induced rotations are mainly due to binding of the drug to D2 receptors that are distributed unequally in both striata. Following apomorphine application, hemi-PD rats and hemi-PD mice rotate to the body side contralateral to the dopamine-depleted hemisphere, which has a higher D2 receptor concentration than the contralateral one [65,70,75]. Acetylcholine is reduced in BoNT-A-injected striata, and thus, the activation of all medium spiny neurons is reduced. Moreover, striatal receptor concentration measurements investigated in BoNT-A-treated hemi-PD rats speak in favor of a BoNT-A-induced reduction of D2 receptors in the respective CPu [22,23].

Considering that the majority of striatal D2 receptors are located on the D2 receptor bearing medium spiny neuron, an unaltered dopamine concentration in the CPu would result in a movement deficit of the contralateral body side via the indirect basal ganglia loop in BoNT-A-treated striata. However, if the majority of striatal D2 receptors are located on the presynaptic terminals of the dopamine afferents from the substantia nigra pars compacta, then dopamine release should be increased due to reduced inhibition by D2 autoreceptors on dopamine terminals [76]. The hypothetically increased striatal dopamine concentration in the BoNT-A-injected CPu would result in an increased movement of the contralateral body side via the indirect basal ganglia loop as suggested by Da Cunha et al. [77]. As all tests for spontaneous motor behavior after striatal BoNT-A revealed an initiation deficit in the contralateral forelimb, the functional significance of D2 autoreceptors for these non-drug-induced behaviors seemed limited.

Since apomorphine is mainly a D2 receptor agonist, its application possibly reverses the alterations in basal ganglia circuitry induced by BoNT-A by shifting the dopamine-mediated functional significance from D2 receptor-bearing medium spiny neuron to D2 autoreceptor-bearing dopamine terminals, thus resulting in a stronger movement initiation in the contralateral body musculature. If dopamine release by the disinhibited dopamine terminals exceeds the apomorphine-induced disinhibition of the D2 receptor-bearing medium spiny neuron, then we can assume the occurrence of a mildly increased contralateral forelimb activity via deactivation of the medium spiny neuron of the indirect basal ganglia loop, which would result in the contralateral rotation behavior [66,67].

#### 3.4.2. Amphetamine-Induced Rotations

Mice with intrastriatal injection of 25 or 50 pg BoNT-A show clear anti-clockwise rotations of about 4–10 per min following the application of amphetamine (Figure 2B). In contrast, comparable experiments in rats revealed no significant effect of BoNT-A injections as compared with vehicle injection (Figure 3B). The obvious difference in BoNT-A-inducible amphetamine rotations between mice and rats is not fully understood.

Amphetamine mainly increases the extracellular dopamine concentration by different mechanisms: it competitively inhibits dopamine uptake via dopamine transporter, facilitates the movement of dopamine from the vesicle into the cytoplasm, and promotes DAT-mediated reverse transport of dopamine into the synaptic cleft independent of action potential-induced vesicular release [78]. Additionally, microdialysis studies in rats revealed amphetamine-induced increased extracellular concentrations of glutamate, aspartate, GABA, taurine, glycine, serotonin, acetylcholine, and of different peptides [79–86].

There is a notable species difference regarding amphetamine-induced contralateral rotation behavior in unilaterally BoNT-A-injected animals, since it is present in mice, but not in rats. Three aspects will be discussed: (1) different effects on axon terminals after intrastriatal BoNT-A injections in mice and rats, (2) the concentrations of the most frequent transmitter receptors in the CPu of naïve mice and rats, and (3) the connectome of the basal ganglia of mice and rats.

Hawlitschka et al. [24] showed that striata from mice and rats react differently after BoNT-A injection with respect to the appearance of TH-ir BoNT-A -induced varicosities [24], since they are consistently found in rats, but were never seen in mice [19,24]. BoNT-A-induced varicosities can be interpreted as a sign of structural alterations induced by BoNT-A. The difference in the occurrence of BoNT-A-induced varicosities between mice and rats might be based on the synaptic vesicle glycoprotein C (SV2C) receptor responsible for the internalization of BoNT-A into the neuron [87,88]. Unfortunately, no data exist concerning differences of SV2C affinity or susceptibility between the CPu of rats and mice [87–95]. It cannot be ruled out that the dopaminergic fibers are differently influenced by BoNT-A and thus the balance or interaction of various transmitter systems on the functional outcome underlying the amphetamine-induced rotation behavior of intrastriatally applied BoNT-A may differ [79,96–101]. In contrast to the interspecies differences in BoNT-A-induced varicosities, ChAT-ir BoNT-A-induced varicosities were consistently found in CPu of both rats and mice following intrastriatal BoNT-A.

#### 3.4.3. Receptors and Connectomics of the CPu

To analyze possible interspecies differences of transmitter systems in the CPu of control rats and mice, we compared the multireceptor fingerprints (Figure S1) of Wistar rats (*n* = 6, data from [23,102, 103]) and of C57Bl/6 mice (*n* = 6; data from [40]). Additionally, the connectome of the mouse basal ganglia generated in a high throughput tract tracing study published as Alan Atlas connectome [104] is compared with rat data (Figure S2). Details to multireceptor fingerprints and connectomes are provided in the Supplement.

#### **4. Conclusions and Future Perspectives**

BoNT-A unilaterally injected into the CPu in naïve mice differentially affected various motor behaviors. Lateralized sensorimotor integration, forelimb preference, and forelimb stepping were significantly impaired contralateral to the injected side. Unilateral intrastriatal BoNT-A induced significant contralateral amphetamine-induced rotations from 1 to 9 months post injection, which is completely opposite to the reaction found in rats. The differences in motor behavior induced by unilateral intrastriatal BoNT-A injections between rats and mice is possibly caused by different effects of BoNT-A on TH-ir fibers in rat and mouse striata, interspecies differences in striatal receptor densities, and different connectomes of the basal ganglia between mice and rats.

Local injection of BoNT-A into the striatum of hemi-PD rats is thought to decrease the local release of acetylcholine therein. Hypercholinism of the striatum caused by acetylcholine released from disinhibited tonically active cholinergic interneurons is held responsible for a disturbed basal ganglia circuitry and, consequently, for motor and behavioral dysfunctions [9,46,48,105]. One possible approach to treat PD is the reduction of the hypercholinism using oral anticholinergic drugs [106,107]. Due to the systemic drug application, adverse side effects such as anticholinergic syndrome and dyskinesia are common [13–15,108]. In the experimental hemi-PD rats, BoNT-A injected locally into

the CPu is beneficial for 3 to 6 months with respect to apomorphine-induced rotational behavior [16–21]. Future experiments will cover two topics: First, repeated intrastriatal BoNT-A injections in hemi-PD rats every 6 months should test whether motor behavior could be improved for a longer time period, which would be a prerequisite for the clinical application of BoNT-A [109–112]. Second, extending our studies of intrastriatal BoNT-A injections to naïve mice, BoNT-A will be injected into mice of various parkinsonian models including those with alterations of relevant PD-associated genes to obtain further insights into PD-related etiology [39,40,113–115].

In conclusion, locally applied BoNT-A, or other botulinum neurotoxins, could be useful in treating brain dysfunctions requiring a deactivation of local brain activity [116]. Advantageously, the effect of local BoNT-A is time-limited and reversible. It can be speculated that following prospective experiments in primates botulinum neurotoxins might be applied as an effective and individually-tailored "chemical neurosurgical approach" [116,117].

#### **5. Materials and Methods**

#### *5.1. Animals*

A total of 46 young adult male C57BL/6 mice (Charles River Wiga, Sulzfeld, Germany) weighing 18–24 g and 28 Wistar rats (strain Crl: WI BR, Charles River Wiga, Sulzfeld, Germany) with a body weight of 280–320 g were used in this study. Animals were housed in standard cages in a temperature-controlled room (22 ± 2 ◦C) under 12 h light/12 h dark conditions with free access to food and water. All procedures were approved by the State Animal Research Committee of Mecklenburg-Western Pomerania (LALLF M-V/TSD/7221.3-1.1-053/08, LALLF M-V/TSD/7221.3-1.1-003/13 from 26 April 2013 and 13 April 2016).

#### *5.2. BoNT-A application*

In the mice, surgery was conducted under aseptic conditions and animals were deeply anesthetized with ketamine (75 mg/kg, bela-pharm Vechta, Germany)/xylazine (5.8 mg/kg, Rompun®, Bayer, Germany), mounted in a mouse adapter (Stoelting, Wood Dale, IL, USA), and fixed in a rat stereotactic apparatus (Kopf, Tujunga, CA, USA). The skull was opened with a dental drill and the mice received an injection of 1 μL BoNT-A solution (Lot No. 13028A1A; List, Campbell, CA, USA, purchased via Quadratech Diagnostics, Surrey, UK) containing a total of 25 pg or 50 pg BoNT-A dissolved in PBS + 0.1% BSA into the right CPu delivered over 4 min using a 26-gauge 5 μL Hamilton syringe, at a rate of 0.25 μL per minute (Figure 5). The sham group received 1 μL BoNT-A vehicle solution. The injection coordinates with reference to bregma were: anterior-posterior = +0.65 mm, lateral = −1.6 mm, and vertical = −3.0 mm from dura, respectively [118] (Figure 5).

Rats were injected either with 1 ng BoNT-A (*n* = 22) or the vehicle solution (sham, *n* = 6) into the right striatum under ketamine (50 mg/kg)/xylazine (4 mg/kg) anesthesia. The BoNT-A solution was injected at two sites, each injection consisting of 0.5 ng BoNT-A solved in 1 μL PBS + 0.1% BSA. Sham-injected rats received the vehicle solution. The coordinates according to bregma were: anterior-posterior = +1.3 mm/−0.4 mm, lateral = −2.6 mm/−3.6 mm and ventral = −5.5 mm/−5.5 mm, respectively [119].

**Figure 5.** (**A**) A coronal Nissl-stained brain section 30 μm thick of a mouse treated intrastriatally with 25 pg BoNT-A 6 months before sacrifice. (**B**,**C**) Higher magnifications of the boxes in (**A**). In (**B**) the needle tract through the cortex is marked by an arrow, in (**C**) the injection channel in the striatum is indicated by two arrows. Scale bar applies to (**A**): 1 mm.

We founded our experiments on the weight values of BoNT-A, i.e., 25 to 50 pg per mouse CPu and 1 ng per rat CPu. In mice the LD50 value after intraperitoneal injection is about 0.25–1.15 ng/kg BoNT-A [120]. Thus, in our experiments a dosage of 50 pg BoNT-A per mouse, injected intrastriatally, is below the classical LD50, and well tolerated. In clinical use the normal amount of BoNT-A (Xeomin, Dysport, Botox and others) is about 200 to 300 units at maximum per patient per session depending on the clinical symptoms and site of application. Remarkably, the clinical efficacy of a "unit" is reported differently by the various manufactures (Botox: 5 ng correspond to 100 units; Dysport: 4.35 ng correspond to 500 units; Xeomin: 0.6 ng correspond to 100 units) [121]. These uncertainties relay on the nonstandardized LD50 bioassays required to achieve the median lethal dose = LD50 (depending on mouse strain, sex, age, volume and route of injection) [122].

#### *5.3. Body Weight*

Body weights were measured before stereotactic surgery and 0.5, 1, 2, 3, 4, 5, 6, 7, and 9 months after injection of BoNT-A or vehicle.

#### *5.4. Behavioral Testing*

Three experimental mouse groups were behaviorally tested: (1) animals receiving 25 pg BoNT-A (25 pg BoNT-A group, *n* = 15), (2) animals receiving 50 pg BoNT-A (50 pg BoNT-A group, *n* = 20), and (3) animals receiving vehicle (sham group, *n* = 11). During the experimental period 5 mice died. Smaller groups were evaluated for the stepping test and the corridor task (25 pg, *n* = 11; 50 pg, *n* = 12; sham, *n* = 10). All mice were adapted to the examination room for 1 h before testing. All tests were performed at 4 time points (1, 3, 6, and 9 months) after the injection of BoNT-A or vehicle, and drug-induced rotations were additionally scored 0.5 months after BoNT-A or vehicle, always according to an identical time schedule (Figure 6). In rats drug-induced rotation tests were carried out 1, 2, 3, and 9 months after BoNT (*n* = 22) or vehicle (*n* = 6) injection.

**Figure 6.** (**A**) Time schedule of the stereotactic application of BoNT-A in mice and behavioral tests. Light grey rectangles symbolize batteries of non-drug-induced behavior tests, dark grey rectangles the subsequently performed apomorphine- and amphetamine-induced rotation tests. (**B**) Detailed visualization of single behavior test batteries. Non-drug-induced tests were performed on the days presented in the light grey part of the time line, drug-induced tests on the days of the dark grey part. The non-drug-induced tests were performed as follows: On the first day the mice underwent the cylinder test until 30 consecutive touches of the glass wall with the forepaws were done. On the second day the stepping test and on the third day the hindlimb clasping were carried out three times per mouse, respectively. On the following 3 days mice underwent food restriction and on the seventh day the corridor task was performed. Following recovery the drug-induced rotation tests were conducted.

#### 5.4.1. Drug-Induced Rotation Tests (Apomorphine, Amphetamine)

Mice rotations were assessed using an automated rotometer system (Rotometer UGO BASILE 43000; Ugo Basile Sri, Gemonio, Varese, Italy). Apomorphine was injected subcutaneously at a dosage of 0.5 mg/kg [123–125] (Teclapharm, Lüneburg, Germany) dissolved in 0.9% sterile saline. Three days later, D-amphetamine sulphate (Sigma Aldrich, München, Germany) was injected intraperitoneally at a dosage of 2.5 mg/kg [124,126,127] (Figure 6). Recording of apomorphineand amphetamine-induced rotations began 5 min after drug injection and lasted 40 min. Rotations were defined as complete 360◦ turns and registered as net difference between the two directions per minute [128]. Anti-clockwise rotations were expressed by positive values, rotations in clockwise direction by negative values. Respective rotations in rats were induced by either 0.25 mg/kg apomorphine applied subcutaneously (Teclapharm, Germany) or 2.5 mg d-amphetamine sulfate (intraperitoneal; Sigma Aldrich, München, Germany), both solved in saline. Rotations were measured over 40 min after apomorphine and over 60 min after amphetamine in a self-constructed rotometer according to Ungerstedt and Arbuthnott [128].

#### 5.4.2. Spontaneous Motor Tests

Mice were tested in the cylinder test, stepping test, hindlimb clasping, and corridor task at 1, 3, 6, and 9 months after injection of BoNT-A or vehicle following a constant time table (Figure 6).

#### Cylinder Test

Forelimb preference was evaluated with the cylinder test as previously described [58,129]. Mice were placed in a glass cylinder (diameter 19 cm, height 20 cm) with mirrors placed behind to allow for a 360◦ view of every contact with the side of the cylinder. Sessions were taped with a video camera system (JVC, GZ-MG255E, Yokohama, Japan), and scored later. For each animal thirty consecutive forepaw contacts with the glass cylinder were evaluated by counting the initial contacts of the right or left paw. Then the ratio of left and right forepaw use was calculated. Evaluation of the videotapes was performed by an observer blinded to the animals' identities.

#### Corridor Task

Lateralized sensorimotor integration and neglect were examined using the corridor task [61]. For our study we used a custom-made 60 cm long, 4 cm wide, and 15 cm high alleyway equipped with 10 pairs of adjacent pots with a diameter of 1 cm, placed at 5 cm intervals and containing 5 sugar pellets (Ain-76A Rodent Tablet 20 mg TestDiet, Richmond, IN, USA). Prior to testing, the mice were food-restricted for three days and maintained at 90% of free-feeding bodyweight during habituation and testing [130]. Animals were adapted to the apparatus for 10 min each on two consecutive days with some scattered sugar pellets along the floor of the corridor and started from different ends of the corridor each day. On the test day, mice were at first positioned in an identical, but empty corridor for 5 min for adaptation and then placed at the end of the testing corridor with bowls containing the pellet. Animals were allowed to move freely along the apparatus for 5 min to retrieve pellets placed on either side of their body. The number of ipsilateral (right side) and contralateral (left side) retrievals made by each mouse was calculated and the data were expressed as a percentage of left and right retrievals of the total number of retrievals. A "retrieval" was defined as a nose poke into a bowl, whether or not pellets were taken, and a new retrieval was counted by investigating a new pot [59,61].

#### Stepping Test

Forelimb akinesia was assessed according to Blume et al. [64] and modified by Heuer et al. [65] using an open table (1.5 m in length). Mice were tested three times in one day during the day light cycle. Each trial was recorded on video. As a first step for habituation to the test, mice were allowed to settle at one end of the table for 1–2 s, with all limbs on the table. Secondly, the experimenter gently lifted up the hindlimbs by pulling up on the tail leaving only the forepaws touching the table surface. Then, at a steady pace of 1 m in 3–4 s the experimenter pulled the animal the total test distance of 1 m backwards by the tail. Finally, the numbers of adjusting steps made with the left and right forepaws were counted offline in the videos.

#### Hindlimb Clasping

Hindlimb clasping is a marker of disease progression in a number of mouse models of neurodegeneration, including certain cerebellar ataxias [131] and parkinsonian mouse models [73,74], and was performed as previously described [72,73]. Outside the cage, each mouse was slowly lifted by the tail for 10 s and then lowered back to the surface. The hindlimbs were observed and the position of the hindlimbs was scored for each trial [72]. A score of 0 indicated that the hindlimbs were consistently splayed outwards and away from the abdomen. A score of 1 indicated that one hindlimb was retracted inwards towards the abdomen more than 50% of the trial period, a score of 2 indicated that both hindlimbs were partially retracted inwards towards the abdomen for at least 50% of the observation period. Mice were tested three times on one day and each trial was recorded on video.

#### *5.5. Receptor Autoradiography and Histology*

The autoradiographic procedure was performed according to standard protocols already published [132–136]. Cryosections of the respective brains were stained with cresyl-violet acetate (SIGMA C1791-5G) to verify the injection sites.

#### *5.6. Statistical Analysis*

Data of all behavioral tests were subjected to two-way ANOVA with repeated measurements. The Holm–Sidak test was used for post-hoc comparisons. The level of significance was set at *p* ≤ 0.05 for all statistical analyses. All statistical tests were done using SigmaPlot 11 Software (Systat Software, Inc., San Jose, CA 95110, USA).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6651/10/7/295/ s1, Figure S1: Multi-receptor fingerprints of the caudate-putamen of mouse (blue) and rat (red), Figure S2: (A) Differential adjacency matrix of the rat and mouse basal ganglia. (B) Differential reciprocity matrix of rat and mouse basal ganglia.

**Author Contributions:** Conceptualization and Supervision, V.A., A.H., O.S. and A.W.; Investigation, A.H., V.A. and A.W.; Visualization, C.H., O.S. and A.H.; Methodology, K.Z., N.P.-G. and T.M.; Writing-Original Draft Preparation , V.A., C.H., O.S., A.W., A.H., N.P.-G. and K.Z.; Writing-Review & Editing, V.A., C.H., A.W., N.P.-G. and K.Z.; Funding Acquisition, A.H., K.Z.

**Funding:** Rostock University Medical Center internal funding FORUN 889005 and 889014, European Union's Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement No 720270 (Human Brain Project SGA1) (K.Z.), and Portfolio Theme "Supercomputing and Modeling for the Human Brain" of the Helmholtz Association.

**Acknowledgments:** We gratefully acknowledge S. Lehmann, F. Winzer, M. Cremer, S. Wilms, S. Buller, J. Bausch, S. Krause and A. Börner for their excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Communication*


**Author details**

Sudarshan Singh1 \*, Tanvi R. Dodiya2 , Rajesh Dodiya3 , Yogesh V. Ushir4 and Slamet Widodo 5

1 Food Technology and Innovation Center of Excellence, Institute of Research and Innovation, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand

2 Parul Institute of Pharmacy and Research, Parul University, Vadodara, Gujarat, India

3 Laxminarayandev College of Pharmacy, Bharuch, Gujarat, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*3.5. Scenario Definition*

To identify which of the seven user groups from the traffic analyses are particularly relevant for the investigation of the synergy effects between e-mobility and PV potential, the potential for the direct use of PV production per user group is examined. For the case study presented here, the user groups "trip for shopping", "trip for execution" and "trip to work" have the highest energy demand in relation to the total energy demand of e-mobility based on the traffic analysis. Since the user groups "trip for shopping" and "trip for execution" have the same characteristic load profile, only the user groups "trip for shopping" and "trip to work" are shown as examples in Figure 9. The figure is used to visualize the determination of the potential for the direct use of PV production for the user groups "trip for shopping" and "trip to work". For the user group "trip to work", the user groups "trip to work with a private car" and "trip to work with a company car" are aggregated due to their similar behaviour. The characteristic of the synthetic charging load profile for the user group "trip to work" is strongly dependent on the sector-specific jobs within the considered area. Therefore, the evening peak, which occurs due to an existing shift operation, can be omitted when looking at another area. The number of peaks for the user group "trip for shopping" is independent of the area under investigation. This means that as soon as there are shopping facilities in the area under consideration, and thus a shopping user group exists, a peak at noon and in the afternoon or evening occurs. In the example shown, around 80% of the energy demand of the user group "trip to work" can be directly covered by PV production, while only 60% can be directly supplied for the user group "trip for shopping".

**Figure 9.** Generic example of a load profiles of the user groups "trip to work" and "trip for shopping" as well as a PV production profile.

The analysis of all user groups indicates that with an increasing number of charging processes during the day, the direct use of the PV production increases and thus the potential. Based on the user behaviour and the share of PV use, the user group "trip to work" can be identified as the user group with the highest potential. Therefore, we have focused on the user groups "trip to work with a private car" and "trip to work with a company car" in combination with a trip chain model to investigate the synergy effects between e-mobility and PV potentials in the case study presented here. Using the trip chain model, the charging energy of the other five user groups is accumulated into these two user groups. Since the number of weekend trips for these user groups is decreasing significantly and it should be ensured that all distances travelled at the weekend are also taken into account, the user group "trip home" is also considered at the weekend.

As already mentioned, the scenarios differ among each other in the penetration of EV and PV. For this purpose, the penetration of EV and PV is varied by 0%, 20%, 40%, 60%, 80% and 100% respectively. In a scenario, all cells have always the same penetration of EV and PV, this means for example that all cells have a penetration of 20% PV and a penetration of 80% EV. This assumption was made, because there are no statistical distributions for urban areas that allow an individual penetration for each cell. Furthermore, two different charging powers (3.7 kW and 11 kW) are considered. For both charging powers, a distinction is made between charging strategy 1 (uncontrolled charging) and charging strategy 2 (controlled charging).

The reference scenario describes the status quo (without PV Potential and future e-mobility) of the medium-voltage grid to be examined. The overview of all simulated scenarios is given in Appendix B, Table A1.


**2. Thyroid hormone**

#### **2.1. Thyroid hormone and thyroid hormone receptors**

Thyroid hormones (THs) include thyroxine (T4) and triiodothyronine (T3). They are synthesized and secreted by the thyroid gland. T4 is the major secreted hormone, while T3 has a higher affinity for TH receptors (TRs). T3 is considered as the active and more potent TH. T4 could be converted to T3 through a deiodination process catalyzed by deiodinases. TH regulates a number of biological functions including growth, development and metabolism in almost all tissues [1]. TH exerts these effects through binding to TRs which are expressed on different cells and tissues. TRs have two isoforms, TRα and TRβ, which are encoded by the THRA and THRB genes, respectively, in humans. Each TR isoform has several splice products, TRα1 (α2) and TRβ1 (β2). TRα1 and TRβ1 are ubiquitously expressed, while TRβ1 is the major TR existed in the liver. TRβ2 is expressed in the hypothalamus, the pituitary gland and the developing brain [2]. TRs are ligand-activated transcription factors, belonging to the family of nuclear receptors (NRs). It can bind to DNA sequences called TH-responsive elements (TREs) together with the retinoid X receptor alpha (RXR-α). In the absence of TH, TRs bind with corepressors, e.g., nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptor (NCOR2), suppressing the transcriptional activity. In the presence of TH, the binding induces a conformational change of TRs, releasing the corepressors and recruiting several co-activators to enhance the transcriptional activity. Since TRs associate with corepressors without ligand binding, it could decrease the transcriptional activity of the target genes. Therefore, it should be cautious to compare the data from animal models in which TRs are genetically deleted with the models with low levels of circulating THs, such as hypothyroidism or thyroidectomy [3].

#### **2.2. Role of TH in cholesterol metabolism**

There is substantial evidence linking TH status with cholesterol or lipid metabolism. Thyroid dysfunction exerts an important effect on the cholesterol level. Hypothyroidism patients typically have elevated plasma cholesterol and increased lipid accumulation in the liver. TH supplement can normalize this lipid dysregulation. THs promote cholesterol synthesis through inducing HMG-CoA reductase and farnesyl pyrophosphate gene expression [1]. THs markedly decrease the expression of apoB-100, the major protein of LDL, while increasing the expression of apo A-I, the major protein of HDL. In addition, THs increase LDLr gene expression. LDLr mediates the uptake of LDL from blood to the liver. Rat LDLr promoter contains two functional TREs. THs could directly bind to the TRE and upregulate the LDLr gene expression [4]. THs may also regulate the clearance of circulating remnant lipoproteins. Hepatic low-density lipoprotein receptor-related protein 1 (LRP1) is a receptor for remnant lipoproteins. Hepatic LRP1 protein expression and function are reduced in the hypothyroidism mouse model. T3 supplement partially normalizes its protein expression level [5]. THs also promote the cholesterol elimination by increasing conversion and secretion of cholesterol into bile acids. In this process, cholesterol 7α-hydroxylase (Cyp7A1), the enzyme in the cytochrome P450 family, is responsible for catalyzing the rate-limiting reaction in the degradation of cholesterol. Cyp7A1 is a direct TR target gene with TREs in its promoter region [6]. ATP-binding cassette (ABC) transporters G5 (ABCG5) and G8 (ABCG8) form a heterodimer that limits intestinal absorption and facilitates biliary secretion of cholesterol. Mice homozygous for disruption of Abcg5 demonstrate a significant reduction in basal biliary cholesterol secretion. T3 treatment does not increase the cholesterol secretion in Abcg5−/− mice as in the wild-type control mice. This observation suggests that THs induce secretion of cholesterol, largely dependent on the ABCG5/G8 transporter complex [7]. THs also modulate gene expression via micro-RNAs. In a human hepatic cell line, THs decrease sterol O-acyltransferase 2 (SOAT2 or ACAT2), the enzyme crucial for the hepatic secretion of cholesterol esters, via miR-181d [8].

T3 also upregulates LDLr gene expression by activating the expression of the sterol regulatory element-binding protein-2 (SREBP-2) and scavenger receptor class B1 (SR-B1) [9]. Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters from HDL to the VLDL and from total triglyceride (TG) to the opposite direction. THs could increase the activity of CETP to influence HDL metabolism [10]. In addition, THs stimulate the lipoprotein lipase (LPL) and hepatic lipase (HL) levels, catabolizing the TG-rich lipoproteins.

## **2.3. Interaction with other transcription factors**

of cholesterol by stimulating the transcription of LDL and HMG-CoA. LDL receptor (LDLr) is responsible for importing LDL from extracellular to intracellular environment for metabolism. Cholesterol is the primary source for biogenesis of steroid hormones. In turn, many hormones exert critical effects on cholesterol synthesis or metabolism. This occurs through the direct effect of these hormones on regulation of the expression or activity of HMG-CoA reductase, SREBP-1c or LDLr. In this chapter, we will discuss the regulatory role of several

Thyroid hormones (THs) include thyroxine (T4) and triiodothyronine (T3). They are synthesized and secreted by the thyroid gland. T4 is the major secreted hormone, while T3 has a higher affinity for TH receptors (TRs). T3 is considered as the active and more potent TH. T4 could be converted to T3 through a deiodination process catalyzed by deiodinases. TH regulates a number of biological functions including growth, development and metabolism in almost all tissues [1]. TH exerts these effects through binding to TRs which are expressed on different cells and tissues. TRs have two isoforms, TRα and TRβ, which are encoded by the THRA and THRB genes, respectively, in humans. Each TR isoform has several splice products, TRα1 (α2) and TRβ1 (β2). TRα1 and TRβ1 are ubiquitously expressed, while TRβ1 is the major TR existed in the liver. TRβ2 is expressed in the hypothalamus, the pituitary gland and the developing brain [2]. TRs are ligand-activated transcription factors, belonging to the family of nuclear receptors (NRs). It can bind to DNA sequences called TH-responsive elements (TREs) together with the retinoid X receptor alpha (RXR-α). In the absence of TH, TRs bind with corepressors, e.g., nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptor (NCOR2), suppressing the transcriptional activity. In the presence of TH, the binding induces a conformational change of TRs, releasing the corepressors and recruiting several co-activators to enhance the transcriptional activity. Since TRs associate with corepressors without ligand binding, it could decrease the transcriptional activity of the target genes. Therefore, it should be cautious to compare the data from animal models in which TRs are genetically deleted with the models with low levels of circulating THs, such as hypothyroidism or thyroidectomy [3].

There is substantial evidence linking TH status with cholesterol or lipid metabolism. Thyroid dysfunction exerts an important effect on the cholesterol level. Hypothyroidism patients typically have elevated plasma cholesterol and increased lipid accumulation in the liver. TH supplement can normalize this lipid dysregulation. THs promote cholesterol synthesis through inducing HMG-CoA reductase and farnesyl pyrophosphate gene expression [1]. THs markedly decrease the expression of apoB-100, the major protein of LDL, while increasing the expression of apo A-I, the major protein of HDL. In addition, THs increase LDLr gene expression. LDLr mediates the uptake of LDL from blood to the liver. Rat LDLr promoter contains two functional TREs. THs could directly bind to the TRE and upregulate

interesting hormones in cholesterol metabolism.

**2.2. Role of TH in cholesterol metabolism**

**2.1. Thyroid hormone and thyroid hormone receptors**

**2. Thyroid hormone**

20 Cholesterol - Good, Bad and the Heart

In addition to the direct action on the cholesterol-related genes, TRs also cross talk with many nuclear receptors to regulate their transcriptions. It shares the same DNA-binding site (direct repeat 4) with liver X receptor (LXR). Activation of TRβ1 by T3 upregulates mouse LXRα, but not LXRβ, mRNA expression in the liver at the transcriptional level [11]. TRβ1 is the major TR mediating the TH effects on plasma cholesterol. ATP-binding cassette transporter A1 (ABCA1) is important for HDL assembly and transporting cholesterol back to the liver for excretion. TR forms a heterodimer with retinoid X receptor (RXR) and binds to the DR-4 element of ABCA1 promoter, suppressing its transcription [12]. The apolipoprotein AV gene (APOA5) is a key determinant of the plasma triglyceride level. It affects the plasma TG level through promoting lipolysis of TG-rich lipoproteins and removal of their remnants [13]. TR-β mediates the effects of THs on the activation of APOA5 gene. Administration of TR-β-selective agonist increases apoAV and diminishes triglyceride levels [14]. In addition, TR-β may compete with LXR/ RXR heterodimers for binding to the DR-4 element in the CYP7A1 promoter [15]. TR-β but not TR-α KO mice completely lost the induction effects of T3 on Cyp7a1 gene, confirming the critical role of TR-β in mediating the TH effect on cholesterol metabolism [16].

Taken together, TH regulates the serum cholesterol level in multiple crucial steps including stimulating its hepatic synthesis, serum uptake and the intrahepatic conversion to bile acids. The physiological level of TH is essential for maintaining the cholesterol homeostasis.


**Potential Anti-SARS-CoV-2 Molecular Strategies**

Editors **Giovanni N. Roviello Caterina Vicidomini**

MDPI ' Basel ' Beijing ' Wuhan ' Barcelona ' Belgrade ' Manchester ' Tokyo ' Cluj ' Tianjin

*Editors* Giovanni N. Roviello Institute of Biostructure and Bioimaging (IBB) Italian National Council for Research (CNR) Naples Italy

Caterina Vicidomini Institute of Biostructure and Bioimaging (IBB) Italian National Council for Research (CNR) Naples Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Molecules* (ISSN 1420-3049) (available at: www.mdpi.com/journal/molecules/special issues/molecules AntiSARSCoV2 Molecular Strategies).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-6963-5 (Hbk) ISBN 978-3-0365-6962-8 (PDF)**

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.


**Contents**

#### **Marila Alfano, Julien P´erard and Christine Cavazza**

## **About the Special Issue Editors**

**Michael J. Maroney** (Professor Emeritus) was born in Ames (Iowa, USA) and received a B.S. in chemistry from Iowa State University in 1977. He received his Ph.D. from the University of Washington Seattle in 1981. Following a short stint at Chevron Research Co., in Point Richmond, CA, he did postdoctoral work at Northwestern University and at the University of Minnesota Minneapolis before joining the faculty at the University of Massachusetts Amherst in 1985.

**Stefano Ciurli** was born in Rosignano Marittimo (Tuscany, Italy) and received a Laurea in chemistry from the University of Pisa (Italy) in 1986, with a thesis carried out at the Department of Chemistry of Columbia University (New York, USA). He received his Ph.D. from Harvard University (Cambridge, MA, USA) in 1990. After two years of postdoctoral studies at the University of Bologna (Italy), he joined the faculty of the University of Bologna in 1992 as an associate professor before becoming a full professor of general and inorganic chemistry in 2001.

## *Editorial* **Bioinorganic Chemistry of Nickel**

**Michael J. Maroney 1,\* and Stefano Ciurli 2,\***

**\*** Correspondence<EMAIL_ADDRESS>(M.J.M<EMAIL_ADDRESS>(S.C.)

Received: 11 October 2019; Accepted: 11 October 2019; Published: 30 October 2019

Following the discovery of the first specific and essential role of nickel in biology in 1975 (the dinuclear active site of the enzyme urease) [1], nickel has become a major player in bioinorganic chemistry, particularly in microorganisms, having impacts on both environmental settings and human pathologies. At least nine classes of enzymes are now known to require nickel in their active sites, including catalysis of redox [(Ni,Fe) hydrogenases, carbon monoxide dehydrogenase, methyl coenzyme M reductase, acetyl coenzyme A synthase, superoxide dismutase] and nonredox (glyoxalase I, acireductone dioxygenase, lactate isomerase, urease) chemistries. In addition, the dark side of nickel has been illuminated in regard to its participation in microbial pathogenesis, cancer, and immune responses. Knowledge gleaned from the investigations of inorganic chemists into the coordination and redox chemistry of this element have boosted the understanding of these biological roles of nickel in each context. In this issue, eleven contributions, including four original research articles and seven critical reviews, will update the reader on the broad spectrum of the role of nickel in biology.

The understanding of the biological role of nickel from the inorganic chemistry side is reviewed on a theoretical basis by Siegbahn et al. [2], who discuss the enzyme mechanisms, including the canonical mechanism of urease, in view of the recently reported crystal structure of the enzyme-substrate complex [3]. This chemistry is further elucidated by original contributions on the pH dependence of binuclear nickel peptide complexes by Keegan et al. [4].

The knowledge of proteins involved in cellular nickel tra fficking (metalloregulators and metallochaperones) is summarized by Higgins in a review [5], which is complemented by a second monographic article by Nim and Wong [6], that focuses more specifically on the maturation of the nickel enzyme urease as a paradigmatic example of how cells balance nickel essentiality and toxicity. These two reviews are augmented by two original research papers on this aspect of the nickel bioinorganic chemistry field: the paper by Alfano et al. [7] is focused on CooJ, an accessory protein necessary for the maturation of the nickel-dependent enzyme carbon monoxide dehydrogenase, while the paper by Barchi and Musiani [8] describes the structure-function relationships in InrS, a nickel-dependent transcription factor from cyanobacteria.

Other reviews in this issue focus on aspects of nickel in human health, with the goal of making this literature more accessible to the bioinorganic community. The general aspects of the field are surveyed by Buxton et al. [9], while a more focused review by Maier and Benoit [10] discusses the role of nickel in microbial pathogenesis. The role of noncoding RNA in nickel-induced human cancer is discussed in a review by Zhu et al. [11], while the role of human acireductone dioxygenase in human health and its metal-dependent function are discussed in the monograph by Liu and Pochapsky [12].

The range of nickel containing systems is still expanding, as demonstrated by the original research paper by Suttisansanee and Honek, which reports a preliminary characterization of a nickel activated and mycothiol-dependent glyoxalase I from fungi [13].

In conclusion, we hope that these open-access contributions will serve as guiding lights for future research into the biological role of nickel. We thank the authors for their original contributions for the special issue, and we thank the reviewers for their insightful comments on each article.


*4.1. The Basic Situation in the BAU Scenario*

The BAU scenario is the benchmark scenario of the paper; that is, almost all results are based on the comparison between the CT/ETS scenario and the BAU scenario. So, the paper needs to report the basic information about the BAU scenario first.

The CO2 emissions in 2060 will be about 16.1 billion tons, increasing by 43% compared with 2020 emissions, which is illustrated in Figure 1. In terms of the primary energy structure, in the BAU scenario in 2060, China's primary fossil energy accounts for 32.0%, and the primary electricity (renewable energy) accounts for 68.0%. The energy structure is much better than the current situation, but there is still a big gap from in carbon neutrality goal.

Because this paper uses a long-term model, we do not consider using Keynesian macro closure conditions but neoclassical macro closure conditions. That is, the factor is fully utilized. Therefore, the main constraints of the whole model come from factor endowment. Thus, in the dynamic process, this paper considers the technological progress and changes in factor endowment (Section 3.2). The changes in capital and labor endowments and technological progress will lead to an increase in GDP. Without carbon constraints, the GDP in the BAU scenario will increase to 739 trillion CNY in 2060, with the primary sectors accounting for 9.3%, the secondary sectors accounting for 25.2%, and the tertiary sectors accounting for 65.5%. In the labor market, the labor population of the primary sectors accounts for 16.9%. Among them, the secondary sectors account for 16.0%, the tertiary sectors account for 65.5%, and the tertiary industry accounts for 67.1%.