3.1. Apoptosis

Apoptosis dysregulation has been suggested to be the underlying cause of several human neurologic diseases. Transient focal or global cerebral ischemia in rodents leads to neuronal apoptosis. Reactive oxygen species (ROS) production is thought to represent a relevant mechanism in the series of biochemical events ultimately leading to apoptosis. CAPE blocks neuronal death through inhibition of inflammation and of mitochondrial cytochrome c release, and through a reduction in free radical generation ^[14][15]. Primary cultures of cerebellar granule neurons (CGNs) are considered a suitable in vitro model to study the mechanisms of neuronal apoptosis, which can be induced by low K⁺ concentrations. CAPE exerts its anti-apoptotic effect on CGNs by blocking ROS formation and inhibiting the activity of both caspase-3 and caspase-9. Caspase-3 is the executioner caspase that is downstream of the intrinsic (mitochondrial) caspase-9 and extrinsic receptor activated (Fas) caspase-8 pathways. Interestingly, CAPE was found to completely block the activation of nuclear factor-κB (NF-κB) without interfering with the marked decrease in intracellular Ca²⁺ concentration induced by low K^{+ [14][16]}.

Another study investigated CAPE’s neuroprotective effect against apoptotic cell death in the developing rat brain after pentylenetetrazole (PTZ)-induced status epilepticus (SE). Prolonged seizures are associated with inadequate blood flow, increased excitatory amino acid release, and decreased glucose use and oxygen consumption. All these features are associated with impaired mitochondrial function and irreversible neuronal damage. CAPE showed diminished apoptosis and preservation of the number of neurons, as well as a reduced number of caspase-3-positive cells in the hippocampus and in the prefrontal cortex ^[17]. Another study ^[18] investigated the effect of CAPE on CGNs exposed to excitotoxicity by the overstimulation of glutamate receptors. Neuronal death caused by excitotoxicity plays an important role in a number of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. CAPE inhibited necrosis mediated by p38 phosphorylation and apoptosis mediated by cytochrome c release and caspase-3 activation, while it appeared to have no effect on glutamate-induced neuronal death via the NF-κB pathway. Interestingly, CAPE proved effective in modulating neurotogenesis—i.e., the formation of new neurites—in PC12 cells challenged with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). CAPE induced the formation, elongation, and ramification of neurites and inhibited the shortage of neurites induced by the neurotoxin. These effects were associated with an increased expression of neuronal typical proteins responsible for axonal growth (GAP-43) and synaptogenesis (synaptophysin and synapsin I) ^[19].

3.2. Neuro-Inflammation

Brain inflammatory events are observed in several neuropathologies or in response to infectious diseases. CAPE’s ability to control neuro-inflammation was compared with that of other drugs, such as acetyl-salicylate (anti-inflammatory), dexamethasone (glucocorticoid), pyrrolidine dithiocarbamate (anti-oxidant), and SN 50 peptide (selective permeant NF-κB inhibitor) in organotypic hippocampal slice cultures. Interestingly, CAPE presented a longer-lasting control (over 48 h after a single administration) of neuro-inflammation compared to the other compounds. This effect can be ascribed to the interference CAPE exerts on several neuroinflammatory effectors, such as NF-κB, tumor necrosis factor (TNF)-α, and nitric oxide (NO). At the dose of maximal effect (100 µM), an increase in cAMP-responsive element binding protein (CREB) activity, a molecule with anti-inflammatory activity, was also observed ^[20]. Another study examining the anti-inflammatory effect of CAPE on activated astroglial cells found that the modulation of NF-κB activity in astroglial cells can be a potential target for the treatment of inflammatory and degenerative CNS diseases. CAPE pre-treatment abrogated the TNF-α induced expression of chemokine (C-C motif) ligand 2 (CCL-2) and intercellular adhesion molecule-1 (ICAM-1) via the inhibition of NF-κB activation in a cell-specific manner. CAPE inhibited the downstream pathways of inhibitor κB (IκB) degradation in monocytic cells while suppressing the activation of upstream IkB kinase in astroglial cells ^[21].

X-linked adrenoleukodystrophy (X-ALD) is a neuro-inflammatory disease associated with the demyelination of the cerebral white matter, characterized by the accumulation of very long chain fatty acids (VLCFA) caused by peroxisomal disorder due to mutations in the ABCD1 gene. CAPE treatment corrected both the metabolic VLCFA accumulation and the secondary inflammation. The effect of CAPE was mediated by the up-regulation of Abcd2 expression and of peroxisomal β-oxidation, leading to decreased VLCFA levels in ABCD1-deficient U87 cells. CAPE administration reduced the expression of inducible nitric oxide synthase (iNOS), inflammatory cytokines, and the activation of NF-κB in primary astrocytes derived by ABCD1/ABCD2-silenced mice ^[22]. Inflammation is a major component in the pathogenesis of another disease, experimental autoimmune encephalomyelitis (EAE), which is considered the animal model of multiple sclerosis (MS). In both EAE and MS, ROS can damage the myelin sheath and the blood–brain barrier (BBB). CAPE maintains cell membrane integrity and function, thus preventing protein leakage and accumulation by inhibiting the peroxidation of membrane lipids. CAPE may exert its anti-inflammatory effect and ameliorate clinical symptoms by inhibiting ROS production at the transcriptional level, through the suppression of NF-κB activation, and by directly inhibiting the iNOS catalytic activity ^[23].

ALS is a disease that causes the death of motor neurons controlling voluntary muscles. In one study in 2009 ^[24], CAPE was selected from a library of 2000 small anti-oxidant molecules for the mechanism of action and the ability to be in the CNS in a concentration sufficient to give a therapeutic effect. In two experimental models of ALS, represented by mouse motor neuron cells (NSC34) expressing mutant superoxide dismutase 1 (SOD1) and motor neurons isolated from cases of familial SOD1-associated ALS, CAPE inhibited NF-κB-induced inflammation. CAPE also activated the nuclear factor erythroid 2-related factor 2 (Nrf2)–ARE pathway, which is usually down-regulated in ALS. Genes up-regulated by Nrf2 include GSH-Px, glutathione reductase, heme oxygenase 1 (HO-1); enzymes involved in GSH synthesis, and NADPH-regenerating enzymes. CAPE effects were tested also in an in vivo model of mice expressing a mutant superoxide dismutase (SOD1^G93A) linked to human ALS. CAPE increased the post-onset survival and lifespan of the mice, showing a significantly greater number of surviving motor neurons and a decreased number of activated microglia and astrocytes in the lumbar region of the spinal cord. In addition, lower levels of phosphorylated p38, a mitogen-activated protein kinase that is involved in both inflammation and neuronal death, were observed in the spinal cords of SOD1^G93A mice ^[25]. Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. CAPE was proven to inhibit cyclooxygenase-2 (COX-2) and iNOS expression and the consequent NO production both in in vitro and in vivo models of microglia activation. Anti-neuroinflammatory responses in microglial cells were mediated by 5′-adenosine monophosphate-activated protein kinase (AMPK)α, erythropoietin (EPO), and HO-1 ^[26].

3.3. Parkinson’s Disease

PD is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons of the substantia nigra pars compacta. 6-Hydroxydopamine (6-OHDA) is a neurotoxic synthetic organic compound used to selectively destroy dopaminergic neurons in the brain to induce PD in laboratory animals. The neurotoxicity and neuronal cell death induced by 6-OHDA are due to ROS production, cytochrome c release, and subsequent caspase-3 activation. CAPE increased the viability of cerebellar granule neurons dose dependently and markedly attenuated the 6-OHDA-induced toxicity. CAPE blocked Ca²⁺-induced cytochrome c release and caspase-3 activation, but no interaction of CAPE with caspase-3 cleavage was found ^[27]. These results were confirmed in the same cellular model and in rostral mesencephalic neurons (RMNs), where CAPE pretreatment increased neuronal viability from 39% to 91% in CGNs and from 33% to 60% in RMNs. CAPE is able to block 6-OHDA-induced dopaminergic neuronal death through the blockage of O²⁻ and peroxynitrite generation, suggesting its potential use as a neuroprotective drug for PD ^[28]. With regard to the in vivo experimental models, CAPE inhibited mitochondrial permeability transition, a process that triggers cytochrome c release and caspase-3 activation, leading to neuronal death in rats treated with 6-OHDA to induce PD. The mechanism of protection involves the scavenging of free radicals and metal chelation without mitochondrial dysfunction ^[29].

CAPE can protect midbrain dopaminergic neurons in vitro and in vivo also by the induction of HO-1 and brain-derived neurotrophic factor (BDNF) expression ^[30]. Rotenone is commonly used to induce experimental PD because of its ability to block the complex I inhibition and to activate microglia, resulting in neuropathologic and phenotypic features of PD. In a rotenone Parkinsonian mice model, CAPE treatment raised the level of striatal dopamine and lessened the inflammatory burden by suppressing microglia cells and down-regulating the gene expression of COX-2, iNOS, and NF-κB. These effects led to an improvement in locomotor activity. CAPE exposure protected against rotenone-induced histopathological abnormalities and led to an increased number of surviving neurons as well ^[31]. In a model for PD induced by chlorpyrifos, CAPE showed positive effects on the paraoxonase (PON1) activity, levels of lipid profile, total sialic acid (TSA), total anti-oxidant capacity (TAC), and total oxidant capacity (TOC) in plasma and brain tissue, preventing neurodegeneration ^[32]. In a murine model of PD induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), CAPE attenuated dopaminergic neurodegeneration and dopamine loss. This effect was associated with a marked reduction in iNOS and caspase-1 expression in vivo. In Vitro, CAPE proved able to mitigate neurotoxicity by inhibiting the mitochondrial release of cytochrome c and apoptosis inducing factor (AIF) induced by MPP^{+ [33]}.

3.4. Alzheimer’s Disease

Alzheimer’s disease (AD) is an age-associated neurodegenerative disease that can be induced in rodents by the intracerebroventricular (ICV) administration of a widely used diabetogenic drug, streptozotocin (STZ). Brain PI3-kinase activity and nitric oxide production mediated by the endothelial NOS (eNOS) are involved in the memory revival function of CAPE in STZ-ICV-administered rats. The basal NF-κB activity in the rat brain was found essential for memory functions as well. The anti-apoptotic and pro-survival functions of PI3-kinase activation regulate eNOS and NFκB activity, conferring neuroprotection and improving memory ^[34]. A previous study by the same group showed that the CAPE down-regulation of oxidative stress and inflammation was accompanied by the amelioration of STZ-ICV-induced dementia. A great increase in the brain GSH levels and a diminution of thiobarbituric acid reactive substances (TBARS) as well as of TNF-α content were observed in the brains of rats treated with CAPE ^[35]. In another study, amyloid-beta oligomers were administered to mice to induce dementia and to study the AD onset and cognitive function impairment. CAPE treatment reversed cognitive deficits and improved learning and memory abilities. This action was accompanied by an induction of Nrf2 and heme oxygenase-1 via the modulation of glycogen synthase kinase 3β in the murine hippocampus ^[36].

3.5. Seizures and Psychosis

CAPE is useful as an adjunctive treatment of seizure disorders. Seizures induced by pentylenetetrazole (PTZ) are due to the activation of glutamate receptors and the inhibition of GABA, an inhibitory neurotransmitter. Glutamate receptors’ activation enhances the ROS level, which in turn enhances glutaminergic activity, but the administration of CAPE increased the latency and decreased the duration of seizures in PTZ-treated mice. CAPE protected the brain tissue from oxidative damage because of its ability to scavenge ROS, to decrease MDA concentration, to increase the anti-oxidant SOD level, and to significantly attenuate NO generation ^[15].

Psychosis has many different causes and can be experimentally induced by dizocilipine maleate (MK-801), which causes neurotoxicity inducing the intracellular generation of free radical species through the N-methyl-D-aspartate (NMDA) receptor blockage. NMDA antagonists mediate cell death triggered by ROS, which are involved in membrane pathology in CNS and play a role in neuropsychiatric disorders, including schizophrenia. In one study focusing on the rat prefrontal cortex (PFC), which is the region mainly affected in schizophrenia, CAPE modulated the brain oxidant/anti-oxidant status, stabilizing the cellular membranous structures. A CAPE therapeutic effect was exerted by decreasing the levels of malondialdehyde, protein carbonyl (PC), and nitric oxide (NO). The activity of the enzymes glutathione peroxidase (GSH-Px), xanthine oxidase (XO), and adenosine deaminase (ADA) in prefrontal tissue were decreased as well, when compared to the MK-801 groups, whereas the catalase activity was not changed. In addition, CAPE treatment decreased the number of apoptotic cells in the PFC exposed to MK-801 ^[37]. Figure 1 summarizes the molecules involved in the effects exerted by CAPE in different neurological conditions described in the previous paragraphs.

Figure 1. CAPE effects on intracellular molecules and pathways in different neurologic disorders.

3.6. Other Diseases

Some pathological conditions, different from neurodegenerative disorders, such as diabetes, septic shock, and hepatic encephalopathy (HE), can affect the health of the nervous system. Diabetes induces oxidative stress and inflammation in brain. In a murine model, CAPE significantly counteracted the effects of diabetes by decreasing the levels of nitric oxide and malondialdehyde and the activities of catalase, glutathione peroxidase, and xanthine oxidase. CAPE treatment also significantly suppressed the expression of inflammatory cytokines such as TNFα and interferon (IFN)-γ and the iNOS activity, which were remarkably enhanced in the brain by diabetes ^[38].

Sepsis patients suffer from severe oxidative stress, with the overproduction of reactive oxygen species and reactive nitrogen species, resulting in direct cellular injury. NF-κB plays a central role in the induction of crosstalk between cytokines and inflammatory mediators, which leads to the pathophysiology of septic shock. The effect of CAPE against lipopolysaccharide (LPS) -induced endotoxemia, neuronal damage, and the associated systemic inflammatory response was investigated in male Wistar rats. CAPE prevented neuronal damage and preserved astrocyte morphology, with no sign of inflammatory cellular infiltration, edema, or cytoplasmic swelling. CAPE decreased the levels of inflammatory cytokines (interleukin (IL)-1α, -1β, -6) and TNF-α in the plasma, increased the anti-inflammatory cytokine levels (IL-4, IL-10), and counteracted the imbalance leading to the inhibition of adhesion molecule expression (sICAM-1). This effect of CAPE on the inflammatory cellular infiltration into the brain could be directly attributed to its inhibitory effect on NF-κB activation ^[39].

Hepatic encephalopathy (HE) is a major neurological complication secondary to severe liver failure, causing serious neurological problems. Thioacetamide-induced HE in rats was almost fully reversed by a combination of CAPE with the laxative lactulose. The survival rates were 37.5% in the HE group, 70% in the HE + lactulose group, 80% in the HE + CAPE group, and 100% in the HE + CAPE + lactulose group. The lack of death in the animals treated with both CAPE and lactulose can be ascribed to the direct neuroprotective effect of CAPE together with the prevention of ammonia production in the body. Increased ammonia, high transaminase levels in blood, increased lipid peroxidation, and decreased antioxidant enzyme activities in most brain regions, along with impaired sensorymotor behavioral tests, were reversed to almost control values in the CAPE + lactulose-treated group ^[40].

Glaucoma is characterized by the death of retinal ganglion cells (RGCs) and visual field defects leading to irreversible blindness. CAPE prevented optic nerve crush-induced RGC apoptosis and neuroinflammation. These effects are mediated by the decreased expression of the inflammatory cytokines IL-8 and IL-6, inducible nitric oxide synthase, cycloooxygenase-2, tumor necrosis factor-α, and chemokine C-C ligand-2. The hypertrophy of astrocytes and Müller cells (gliosis) was modulated by CAPE through the inhibition of NF-κB signaling ^[41].