Doxycycline inhibits dopaminergic neurodegeneration through upregulation of axonal and synaptic proteins.

Doxycycline (DOX) is a widely used antibiotic that is able to cross the blood-brain barrier. Several studies have shown its neuroprotective effect against neurodegeneration and have associated it with antioxidant, anti-apoptotic, and anti-inflammatory mechanisms. We have recently demonstrated that DOX mimics nerve growth factor (NGF) signaling in PC12 cells. However, the involvement of this mechanism in the neuroprotective effect of DOX is unknown. Axonal degeneration and synaptic loss are key events at the early stages of neurodegeneration, and precede the neuronal death in neurodegenerative diseases, including Parkinson's disease (PD). Therefore, the regeneration of the axonal and synaptic network might be beneficial in PD. The effect of DOX in PC12 cells treated with the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) was addressed. Doxycycline reduced the inhibition of neuritogenesis induced by MPP+, even in cells deprived of NGF. The mechanism involved the upregulation of GAP-43, synapsin I, ß-III-tubulin, F-actin, and neurofilament-200, proteins that are associated with axonal and synaptic plasticity. Considering the role of axonal degeneration and synaptic loss at the initial stages of PD, the recent advances in early diagnosis of neurodegeneration, and the advantages of drug repurposing, doxycycline is a promising candidate to treat PD.

The antibiotic doxycycline mimics the NGF signaling in PC12 cells: A relevant mechanism for neuroprotection.

Doxycycline has been used as antibiotic since the 1960s. Recently, studies have shown that doxycycline is neuroprotective in models of neurodegenerative diseases and brain injuries, mainly due to anti-inflammatory and anti-apoptotic effects. However, it is not known if doxycycline has neurotrophic potential, which is relevant, considering the role of axonal degeneration at the early stages of neurodegeneration in Alzheimer's disease, Amyotrophic Lateral Sclerosis and Parkinson's disease as well as in normal aging. Axons are preceded by the formation of neurites, the hallmark of the neuronal differentiation induced by neurotrophins like NGF. Therefore, the modulation of neurotrophin receptors aimed at formation and regeneration of axons has been proposed as a strategy to delay the progression of neurodegeneration and has gained relevance as new techniques for early diagnosis arise. Based on these premises, we investigated the potential of doxycycline to mimic the effects of Nerve Growth Factor (NGF) with focus on the signaling pathways and neuronal modulators of neurite initiation, growth and branching. We used PC12 cells, a neuronal model widely employed to study the neurotrophic pathways and mechanisms induced by NGF. Results showed that doxycycline induced neurite outgrowth via activation of the trkA receptor and the downstream signaling pathways, PI3K/Akt and MAPK/ERK, without inducing the expression of NGF. Doxycycline also increased the expression of GAP-43, synapsin I and NF200, proteins involved in axonal and synaptic plasticity. Altogether, these data demonstrate, for the first time, the neurotrophic potential of doxycycline, which might be useful to restore the neuronal connectivity lost at the initial phase of neurodegeneration.

The Neurotrophic-Like Effect of Carvacrol: Perspective for Axonal and Synaptic Regeneration.

Carvacrol (CARV) is a phytochemical widely used as flavoring, preservative, and fragrance in food and cosmetic industries. CARV is able to cross the blood-brain barrier (BBB) and has demonstrated protective potential against neurodegenerative diseases by several mechanisms, including antioxidant, anti-inflammatory, anticholinesterase, and antiapoptotic effects. However, it is not known whether CARV is able to modulate axonal and synaptic plasticity, crucial events in cognition, memory, and learning. Abnormalities in axonal and synaptic plasticity, low levels of neurotrophins, and bioenergetic failure have been associated with the pathogenesis of neurodegenerative diseases, including Parkinson's (PD) and Alzheimer's diseases (ADs). Small lipophilic molecules with neurotrophic activity might be able to restore the axonal and synaptic networks that are lost in neurodegenerative processes. Therefore, this study investigated the neurotrophic potential of CARV in PC12 cell-based neuronal model. Carvacrol induced neurite outgrowth by activating the NGF high-affinity trkA receptor and the downstream PI3K-AKT and MAPK-ERK pathways, without depending on NGF. In addition, CARV increased the expression of proteins involved in neuronal plasticity (ß-tubulin III, F-actin, 200-kDa neurofilament, GAP-43 and synapsin-I) and improved bioenergetics (AMPKα, p-AMPKα, and ATP). Our study showed, for the first time, a promising neurotrophic mechanism of CARV that could be beneficial in neurodegenerative and neurological diseases.

Caffeic Acid Phenethyl Ester (CAPE) Protects PC12 Cells Against Cisplatin-Induced Neurotoxicity by Activating the AMPK/SIRT1, MAPK/Erk, and PI3k/Akt Signaling Pathways.

Peripheral sensory neuropathy (PSN) is a well-known side effect of cisplatin characterized by axonal damage. In the early stage of neurotoxicity, cisplatin affects proteins that modulate neurite outgrowth and neuroplasticity, without inducing mitochondrial damage or apoptosis. There are no preventive therapies for cisplatin-induced peripheral neuropathy; therefore, measures to improve axonal growth and connectivity would be beneficial. Caffeic acid phenethyl ester (CAPE) is a bioactive component of propolis with neurotrophic and neuroprotective activities. We have recently showed that CAPE protects against cisplatin-induced neurotoxicity by activating NGF high-affinity receptors (trkA) and inducing neuroplasticity. We have now assessed other potential early targets of cisplatin and additional mechanisms involved in the neuroprotection of CAPE. Cisplatin reduced axonal cytoskeletal proteins (F-actin and ß-III-tubulin) without inducing oxidative damage in PC12 cells. It also reduced energy-related proteins (AMPK α, p-AMPK α, and SIRT1) and glucose uptake. At this stage of neurotoxicity, glutamate excitotoxicity is not involved in the toxicity of cisplatin. CAPE attenuated the downregulation of the cytoskeleton and energy-related markers as well as SIRT1 and phosphorylated AMPK α. Moreover, the neuroprotective mechanism of CAPE also involves the activation of the neurotrophic signaling pathways MAPK/Erk and PI3k/Akt. The PI3K/Akt pathway is involved in the upregulation of SIRT1 induced by CAPE, but not in the upregulation of cytoskeletal proteins. Altogether, these findings suggest that the neuroprotective effect of CAPE against cisplatin-induced neurotoxicity involves both (a) a neurotrophic mechanism that mimics the mechanism triggered by the NGF itself and (b) a non-neurotrophic mechanism that upregulates the cytoskeletal proteins.

The cannabinoid beta-caryophyllene (BCP) induces neuritogenesis in PC12 cells by a cannabinoid-receptor-independent mechanism.

Beta-caryophyllene (BCP) is a phytocannabinoid whose neuroprotective activity has been mainly associated with selective activation of cannabinoid-type-2 (CB2) receptors, inhibition of microglial activation and decrease of inflammation. Here, we addressed the potential of BCP to induce neuritogenesis in PC12 cells, a model system for primary neuronal cells that express trkA receptors, respond to NGF and do not express CB2 receptors. We demonstrated that BCP increases the survival and activates the NGF-specific receptor trkA in NGF-deprived PC12 cells, without increasing the expression of NGF itself. The neuritogenic effect of BCP in PC12 cells was abolished by k252a, an inhibitor of the NGF-specific receptor trkA. Accordingly, BCP did not induce neuritogenesis in SH-SY5Y neuroblastoma cells, a neuronal model that does not express trkA receptors and do not respond to NGF. Additionally, we demonstrated that BCP increases the expression of axonal-plasticity-associated proteins (GAP-43, synapsin and synaptophysin) in PC12 cells. It is known that these proteins are up-regulated by NGF in neurons and neuron-like cells, such as PC12 cells. Altogether, these findings suggest that BCP activates trka receptors and induces neuritogenesis by a mechanism independent of NGF or cannabinoid receptors. This is the first study to show such effects of BCP and their beneficial role in neurodegenerative processes should be further investigated.

The neuroprotection of cannabidiol against MPP⁺-induced toxicity in PC12 cells involves trkA receptors, upregulation of axonal and synaptic proteins, neuritogenesis, and might be relevant to Parkinson's disease.

Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP(+) toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP(+), probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP(+), a neurotoxin relevant to Parkinson's disease.

Caffeic acid phenethyl ester (CAPE) protects PC12 cells from MPP+ toxicity by inducing the expression of neuron-typical proteins.

Neurite loss is an early event in neurodegenerative diseases; therefore, the regeneration of the network of neurites constitutes an interesting strategy of treatment for such disorders. Neurotrophic factors play a critical role in neuronal regeneration, but their clinical use is limited by their inability to cross the blood brain barrier. Oxidative and inflammatory events are implicated in neurodegeneration and antioxidant compounds have been suggested as potential neuroprotectors. The protective potential of CAPE (caffeic acid phenethyl ester) has been shown in different models of neurotoxicity (in vitro and in vivo) and it has been associated with immune-modulatory, antioxidant and anti-inflammatory properties; however, other mechanisms might be involved. The present study demonstrates that CAPE protects PC12 cells from the cellular death induced by the dopaminergic neurotoxin MPP(+) by increasing the network of neurites. Results showed that CAPE induced the formation, elongation and ramification of neurites in PC12 cells non-stimulated with NGF (nerve growth factor) and inhibited the shortage of neurites induced by the dopaminergic neurotoxin. These effects were associated with increased expression of neuron-typical proteins responsible for axonal growth (GAP-43) and synaptogenesis (synaptophysin and synapsin I). It is noteworthy that, unlike neurotrophins, CAPE would be able to cross the blood brain barrier and exert its neurotrophic effects in the brain. This study corroborates the therapeutic potential of CAPE in neurodegenerative diseases while proposes the involvement of neuroplasticity in the mechanism of neuroprotection.