2. The Role of SAHA as a Neuroactive Compound
Neuroprotective effects refer to the ability of a compound to protect neurons from damage or degeneration, thereby preserving their structure and function. SAHA has been demonstrated to have the potential to exert such neuroprotective effects, which could be beneficial in preventing cognitive decline and maintaining cognitive function. Broader assessments of in vivo and in vitro treatments have shown that this activity has multiple downstream effects and is not restricted to a particular signaling pathway (Table 1).
Table 1. Molecular functions of SAHA as a neuroactive compound.
Additionally, SAHA has shown neuritogenic effects, meaning it can promote the growth and development of neurites. Neurites are essential for establishing neural connections and facilitating proper communication between neurons. By enhancing neurite growth, SAHA could potentially aid in the restoration of neuronal connectivity, which may be disrupted in cognitive impairment and epilepsy.
By enhancing neurite growth, SAHA could potentially aid in the restoration of neuronal connectivity, which may be disrupted in cognitive impairment and epilepsy. The combination of both neuroprotective and neuritogenic effects of SAHA strengthens its potential for drug repurposing in the treatment of cognition impairment and epilepsy.
2.1. Neuronal Maturation and Plasticity
Morphological and molecular data indicate that SAHA-treated ESCs are committed toward neural differentiation with a dose-dependent effect that is cytotoxic at higher doses and pro-proliferative at lower concentrations 
. Furthermore, it has been shown that SAHA promotes a significant increase in mean neurite length, number of neurites/cell, and neurite length/cell via activation of the ERK pathway 
. This activity is extremely interesting because in some instances ERK activation may lead to detrimental effects and contribute to neurodegeneration; in other cases, it fosters survival and differentiation or counteracts pathogenic effects of mutations in Huntington’s disease models (HD). Indeed, several studies have reported the dual role of ERK1/2 signaling, as a double-edged sword 
, that in response to specific signals may lead to beneficial effects on long-term and short-term memory formation and neuronal protective activity in Huntington’s disease models (HD) 
and to detrimental effects that induce neurotoxicity 
. This double role of ERK1/2 signaling may reflect differences in the functional response to SAHA, depending on target cells and tissue, as well as the dosage used 
Supporting this evidence of SAHA as a proneuronal molecule, researchers previously showed the activity of this epidrug counteracting the defective neuronal differentiation and maturation in animal and cellular models ablated for aristaless homeobox-related gene (ARX
), a well-conserved disease gene involved in multiple NDDs 
. Specifically, SAHA is able to recover KDM5C-H3K4me3 signaling and improve neuronal differentiation both in Arx-KO murine ES-derived neurons and ARX/alr-1-deficient Caenorhabditis elegans
animals. Behavioral phenotype analysis of alr-1-KO worm mutants revealed that SAHA rescued defective touch response, a behavioral defect affecting mechanosensory neurons 
. Consistent with these findings, it has been shown that SAHA stimulates the expression of BDNF in cultured rat neurons 
and controls BDNF release in different brain cell populations contributing to memory formation 
. Furthermore, several investigations proved the association of SAHA with long-lasting chromatin modifications regulating the expression levels of plasticity-related genes or involved in signaling networks that regulate neuronal morphogenesis 
. Noteworthy, in human iPSC-derived neurons and mouse embryonic cortical cells, SAHA upregulates—in a dose-dependent manner—the expression of evolutionarily conserved gene networks with key roles in synaptic maintenance and function 
. Further studies are required to establish in detail the molecular benefits, as well as to balance the possible installation of inappropriate histone modifications.
2.2. Activation of Autophagy
Emerging evidence suggests that neuronal autophagy regulates the refinement of the polarized morphology of neurons (e.g., axonal outgrowth, dendritic complexity, and spine pruning) and the formation and maintenance of synapses. Mechanistically, it is a complex catabolic process that delivers cytosolic components to lysosomes for degradation. SAHA has been shown to influence the autophagy pathway at various points and to protect the aged brain against plasticity impairment. This is what emerges from two recent studies showing that cognitive dysfunction induced by the anesthetic sevoflurane or insulin resistance can be counteracted by SAHA, through the expression control of autophagy-related gene markers, such as LC3 and P62 
. As for the mechanism of action, previous studies carried out in MEF cells demonstrated that SAHA induces autophagy by activating the ULK1 complex, which is the most upstream component in the core autophagy pathway, and by suppressing the mammalian target of rapamycin (mTOR), which in turn inhibits autophagy induction 
. Moreover, several studies have shown that perturbations affecting autophagy may cause NDDs. For example, mutations in genes encoding regulatory autophagic proteins have been detected in ASD patients characterized by elevated mTOR activity (mTORopathy) and defective autophagy 
. Elevated mTOR signaling activity has been also detected in patients with fragile X syndrome 
and in congenital forms of epileptogenesis and cortical malformations 
. Given the activity of SAHA as an mTOR inhibitor, this epidrug might be a promising therapy to correct neuronal autophagy in mTOR-related NDDs. However, further studies are required to identify the autophagy-related pathways involved in neurodevelopmental processes in order to identify an appropriate time window for SAHA administration.
2.3. Microtubule Organization
Microtubules (MTs) are cylindrical cytoskeletal structures constituted by polymers of αβ-tubulin that play essential roles in many essential mammalian cell functions, such as cytoskeletal support and transportation of intracellular cargo, thus regulating neuroplasticity and synaptogenesis. The dynamic changes regulating MT structure and stability are mediated by acetylation of K40 in α-tubulin, a posttranslational modification, which marks the luminal surface of microtubules and is fundamental for microtubule stabilization and vesicle transportation. The dynamics between acetylation/deacetylation of α-tubulin is mediated by the action of acetyltransferases and deacetylases, respectively. Because of its HDACi activity, SAHA induces hyperacetylation of α-tubulin, facilitating sliding between filaments and making MTs more mechanically stable 
. A defective level in the α-tubulin acetylation has been detected in animal models for different neurological disorders, including cortical malformations 
, Rett syndrome 
, and various neurodegenerative pathologies 
. For some of these pathologies, treatments with other HDAC inhibitors rescue the defective α-tubulin acetylation and thus correct a number of MT abnormalities, such as the defective axonal transport observed in Charcot–Marie–Tooth primary neurons 
and the aberrant BDNF vesicle trafficking observed in Rett patient fibroblasts 
. Taking this evidence into account, the applicability of SAHA to counteract cytoskeleton malfunctioning in NDDs seems to be very promising to be addressed.
Related to the acetylation/deacetylation effect of SAHA, Luo et al. (2022) underlined a new mechanism of action preventing axonal damage and neurological dysfunction in a rat model of subarachnoid hemorrhage (SAH). This SAHA-induced response is mediated by heat shock protein (HSP) co-inducer activity via the acetylation of HSP70, which in turn induces the degradation of TDP-43 aggregates 
2.4. Regulation of Alternative Splicing Switch
Alternative splicing (AS) is a critical process of posttranscriptional gene expression that diversifies and expands the proteome and increases the functional diversity of molecules implicated in mammalian brain development and plasticity 
. Characterized by high flexibility, AS is a complex mechanism controlled via RNA-binding proteins (RBPs) forming a megadalton complex named spliceosome 
In relation to the levels of histone acetylation, the opening and closing states of chromatin have the ability to influence exon inclusion and exclusion by recruiting directly splicing factors to sites of specific exons 
. Indeed, histone hyperacetylation, which creates a more open chromatin structure, leads to a faster elongation rate phase allowing for the recruitment of splicing factors to a strong splicing site, resulting in exon skipping. Conversely, a slower elongation rate can recruit splicing factors to weak upstream splice sites, resulting in exon inclusion 
. In these frameworks, splicing defects can be overcome by manipulating HDACs. In addition to that, a recent study proved that SAHA is also capable to counteract splicing abnormalities by rescuing RNA foci, as demonstrated in a mouse model of myotonic dystrophy type 1 (DM1) 
. DM1 is the most common form of adult neuromuscular dystrophy caused by the abnormal expansion of CTG repeats in the 3′ untranslated region of the dystrophia myotonic protein kinase (DMPK
) gene. These expanded repeats lead to the formation of hairpin structures affecting the DMPK
mRNA (GC-rich foci), which in turn causes the sequestration of splicing regulators such as MBLN1, resulting in aberrant alternative splicing of a variety of mRNAs 
. The authors proved that SAHA is able to reduce RNA foci formation.
More interestingly, the daily intraperitoneal injections of SAHA in DM1 mice and the daily administration in DM1 patient-derived myoblasts counteract the aberrant splicing affecting myotonia genes (e.g., chloride channel genes) 
. Moreover, Nakano et al. 
proved the efficacy of SAHA to counteract aberrant gene expression caused by the defective splicing of the RE1 silencing transcription factor (REST) gene. This gene encodes a transcriptional repressor that forms a protein complex with HDAC1 and HDAC2. Since HDACs are critical for REST-dependent gene expression, the authors demonstrated that the subcutaneous injection of SAHA in Rest+/ΔEx4
mice induces histone hyperacetylation, rescues defective gene expression of REST target genes, and subsequently induces improvement of cochlear defects