Therapeutic advances in neural regeneration for Huntington’s disease

Francesco D’Egidio, Vanessa Castelli, Giorgia Lombardozzi, Fabrizio Ammannito, Annamaria Cimini, Michele d’Angelo

Abstract Huntington’s disease is a neurodegenerative disease caused by the expansion mutation of a cytosineadenine-guanine triplet in the exon 1 of the HTT gene which is responsible for the production of the huntingtin (Htt) protein.In physiological conditions, Htt is involved in many cellular processes such as cell signaling, transcriptional regulation, energy metabolism regulation, DNA maintenance,axonal trafficking, and antiapoptotic activity.When the genetic alteration is present, the production of a mutant version of Htt (mHtt) occurs, which is characterized by a plethora of pathogenic activities that, finally, lead to cell death.Among all the cells in which mHtt exerts its dangerous activity, the GABAergic Medium Spiny Neurons seem to be the most affected by the mHtt-induced excitotoxicity both in the cortex and in the striatum.However, as the neurodegeneration proceeds ahead the neuronal loss grows also in other brain areas such as the cerebellum, hypothalamus,thalamus, subthalamic nucleus, globus pallidus, and substantia nigra, determining the variety of symptoms that characterize Huntington’s disease.From a clinical point of view, Huntington’s disease is characterized by a wide spectrum of symptoms spanning from motor impairment to cognitive disorders and dementia.Huntington’s disease shows a prevalence of around 3.92 cases every 100,000 worldwide and an incidence of 0.48 new cases every 100,000/year.To date, there is no available cure for Huntington’s disease.Several treatments have been developed so far, aiming to reduce the severity of one or more symptoms to slow down the inexorable decline caused by the disease.In this context, the search for reliable strategies to target the different aspects of Huntington’s disease become of the utmost interest.In recent years, a variety of studies demonstrated the detrimental role of neuronal loss in Huntington’s disease condition highlighting how the replacement of lost cells would be a reasonable strategy to overcome the neurodegeneration.In this view, numerous have been the attempts in several preclinical models of Huntington’s disease to evaluate the feasibility of invasive and non-invasive approaches.Thus, the aim of this review is to offer an overview of the most appealing approaches spanning from stem cell-based cell therapy to extracellular vesicles such as exosomes in light of promoting neurogenesis, discussing the results obtained so far, their limits and the future perspectives regarding the neural regeneration in the context of Huntington’s disease.

Key Words: cell therapy; exosomes; extracellular vesicles; Huntingtin; Huntington’s disease; medium spiny neurons; neurodegenerative disease; neurogenesis; neuronal loss; stem cells

Huntington’s Disease: an Overview

Huntington’s disease (HD) is a fully penetrant neurodegenerative disease caused by the mutation of a single gene: an expansion mutation of a cytosineadenine-guanine (CAG) triplet in the huntingtin gene (HTT) exon 1.TheHTTgene is located in the short arm of chromosome 4 (4p.16.3) and it is responsible for the production of the huntingtin (Htt) protein.To date, the physiological activity of Htt is not fully understood.Decades of research showed the involvement of Htt in many cellular processes such as cell signaling, transcriptional regulation, energy metabolism regulation, DNA maintenance, axonal trafficking, and antiapoptotic activity.Nevertheless,when the expansion in the exon 1 of theHTTgene occurs a mutant Htt(mHtt) characterized by a prolonged polyglutamine (polyQ) tract is produced.The expanded polyQ tract prevents the proper fold of mHttthat remains as unfolded soluble protein able to aggregate into oligomers, with the formation of mHtt large inclusions and fibrils both in the cytoplasm and nucleus.Moreover, recent studies suggested a toxic activity for N-terminal mHtt oligomers produced by aberrant splicing responsible for the fabrication of a short mRNA which is translated into a toxic N-terminal fragment containing the exon 1 but also by the proteolytic cleavage of the aberrant protein.In this view, mHtt can affect all the activities in which the wild-type Htt is involved, leading to an overall cellular dysfunction that ends in HD pathology.Among all the features characterizing HD at cellular and molecular levels, it is possible to find oxidative stress, neuroinflammation, excitotoxicity, altered vesicle trafficking, organelles, and damaged proteins clearance system dysfunction, reduced neurotrophins levels, mitochondrial impairment, as well as altered nuclear pore complex, altered DNA methylation and transcriptional deregulation (Andhale and Shrivastava, 2022).

HD shows a prevalence of around 3.92 cases every 100,000 worldwide and an incidence of 0.48 new cases every 100,000/year, with 7.1% of all the new cases represented by de novo mutations (Medina et al., 2022).The causative mutation of the trinucleotide expansion is dominantly inherited.A correlation between the number of CAG repeats and the advent of symptoms exists in HD.In fact, individuals with 36 or more repeats will surely manifest HD’s characteristics, even if between 36 and 39 repeats the penetrance is reduced.Above 36 CAG repeats, greater numbers of triplets are related to earlier onset of HD.Moreover, a higher risk can be observed in children with a paternally inherited polyQ tract of intermediate range of length, leading to the anticipation phenomenon due to a pathogenic expanded polyQ tract.The reason behind this feature relies on the greater instability of the CAG repeat in male sperm than in somatic tissues (Wheeler et al., 2007).To assess the presence and the entity of the mutation in people at risk, a predictive genetic test can be performed.Nevertheless, diagnosis by genetic analysis is not the only possible way to establish the presence of HD features.From a clinical point of view, HD is characterized by a wide spectrum of symptoms, among which cognitive disorders, motor impairment, and dementia can be found as predominant signs.After years, the symptoms getting worse lead to an overall dysfunction ending with the death of the HD patient, usually in 15–20 years(Ghosh and Tabrizi, 2018; D’Egidio et al., 2023).

Neurodegeneration and symptoms in HD

Neurodegeneration seems to affect patient’s brains in a specific order of events.In the first phase of HD striatum and cortex show signs of neuronal loss, with the striatum being more vulnerable to mHTT than the cortex.Then, neurodegeneration occurs in the cerebellum, hypothalamus, thalamus,subthalamic nucleus, globus pallidus, and substantia nigra, as the disease progresses, with neuropathological features involving temporal and frontal lobes in the advanced phases of HD.In the degeneration process, among all the neurons damaged, GABAergic medium spiny neurons (MSNs) that are predominantly present in the striatum and less abundant in the cortex show higher vulnerability to the excitotoxicity that characterizes HD (Andhale and Shrivastava, 2022).As the neuronal loss gets worse spreading around the brain, the outcomes lead to the manifestation of signs and symptoms that characterize HD.For instance, motor symptoms appear as the MSN degeneration worsens in the striatum.The impairment of the motor behavior manifests as physical instability, difficulty in mastication, swallowing and speaking, and rigidity, but mainly in out-of-control and unintentional choreic movements.The motor features are peculiar in HD due to the imbalance between the indirect and the direct pathway related to the receptors 1 (D1R)and 2 (D2R) for the dopamine neurotransmitter.The receptors are expressed by MSNs mainly in the limbic system, thalamus, hypothalamus, and striatum.The result of the MSN degeneration in the early phase of HD is hyperkinesia,as the levels of D2R decrease causing the over-activation of the motor cortex with the derangement of the indirect pathway.In the later phases of HD, the hyperkinesia switches to hypokinesia.In fact, the lower levels of D1R in the later phases of the disease are responsible for the disruption of the direct pathway via the thalamus, and the over-inhibition of the motor cortex.Thus,as the degeneration of MSNs progresses individuals with HD will experience the switch between hyperkinesia and hypokinesia, ending in rigidity in the final stages of HD.The degeneration responsible for the motor impairment that involves the striatum and the hippocampal region of the frontal cortex is, moreover, responsible for cognitive symptoms (Goodliffe et al., 2018;Bergonzoni et al., 2021).In this regard, impairment in flexibility, abstract thinking, rule acquisition, and judgment are related to subcortical atrophy and the disruption of the frontal-subcortical circuitry.Finally, behavioral symptoms and mood changes occur as the degeneration reaches the cingulate cortex causing depression, irritability, anxiety, anger, and compulsive behavior.As the symptoms appear, they inexorably get worse, till death reaches the patients(Abdollah Zadegan et al., 2023; Ruiz-Idiago et al., 2023).

To date, there is no available cure for HD.However, the interdisciplinary management of symptoms seems to be an efficacious road to counteract signs of HD and to ameliorate the quality of life of affected people.Interestingly, in this context, given the role of neurodegeneration, several studies deepened the knowledge around cellular and tissue regeneration, proposing reliable strategies to counteract neuronal loss and restore the brain of people with HD.Therefore, this review aims to offer an overview of the knowledge around regenerative research and HD, highlighting the most innovative approaches proposed in the past and recent literature in light of neurogenesis as the common denominator.

Data Source

Extensive bibliographic research was conducted using the PubMed National Library of Medicine (NIH), Web of Science platform, Google Scholar, and Clinical Key databases.Examples of the search terms used were “Huntington’s disease”, “neurodegeneration”, “neurogenesis”, “regeneration”, “adult neurogenesis”, “extracellular vesicles”, “cell therapy”, “neurotrophic factors”,“BDNF”, “NGF”, “GDNF”, “therapeutic approach”, “in vitro”, “in vivo”, “clinical study”, “preclinical study”, “experimental studies”.For screening, a restriction was made to those papers published in the last 5–7 years in English.Priority was given to prospective studies and reviews with adequate methodological quality.In addition, a secondary search of the bibliography of the papers finally selected was conducted to detect possible omissions.For the analysis of all relevant publications, consensus meetings were held with all the authors.

Neurogenesis and Huntington’s Disease

The interplay of all the pathological features of HD is responsible, in the end,for a common result, neuronal loss.In this peculiar context, the brain tries to adapt to and counteract the stressful condition caused by HD in a plethora of modalities, among which is the neurogenesis process.In particular, the adult brain of mammalians shows the ability to produce new neurons (Doetsch,2003).So far, this process has been observed in specific areas of the brain,mainly in the subventricular zone (SVZ) of the lateral ventricles as well as in the subgranular zone of the dentate gyrus of the hippocampus.New progenitor cells, neuroblasts, are generated by neural stem cells in the adult SVZ (Jurkowski et al., 2020; Cebrian-Silla et al., 2021).The neuroblasts, as committed cells, migrate to the olfactory bulb via the rostral migratory stream.Here, the immature neurons detach from the rostral migratory stream and move to the glomeruli where they differentiate into functional interneurons,such as periglomerular and granule neurons.The most abundant part of interneurons become GABAergic granule neurons characterized by dendrodendritic synapses formed with tufted and mitral cells and by the absence of axons.Regarding periglomerular neurons, they represent the smaller part of the cited above interneurons and are mainly GABAergic neurons,even if a small part is dopaminergic too.From the adult subgranular zone of the dentate gyrus, the proliferation of non-radial and radial progenitors is responsible for the generation of intermediate progenitors from which new neuroblasts are produced.The new neuroblasts move to the inner granule zone where they differentiate into dentate granule cells, integrating themselves into the local circuitry (Lois et al., 1996; Doetsch et al., 1997,1999).Their integration begins with the tonic stimulation produced by the GABA released in the extracellular space by local interneurons.At this point, GABAergic, at first, and glutamatergic synaptic inputs occur, followed by synaptic outputs of mossy fibers to CA3 and hilar neurons (Ming and Song, 2011).The newborn adult neurons show increased synaptic plasticity compared to other mature cells, such as mature granule cells.Moreover,these neurons show hyper-excitability till a prolonged maturation phase reaches the end.After this phase, the mature adult-born neurons manifest electrophysiological properties close to mature neurons showing, for instance,firing behavior and kinetics and amplitude of glutamatergic and GABAergic inputs.Even if the milestones of development are conserved among adult neurogenesis and embryonic early postnatal neurogenesis, the maturation of neurons is slower in adult development compared to the embryonic one, with faster maturation ending in new neurons aberrantly integrated into the adult hippocampus (Bond et al., 2022).In this context, newborn adult neurons have been proposed to have a role in cognition, given the involvement of the newly integrated neurons in the hippocampal circuitry and functioning.In fact, the hippocampus implication in learning and memory highlights the relevance of adult neurogenesis in modulating memory formation suggesting its possible participation in the cognition process (Lieberwirth et al., 2016; Dard et al.,2019).For this reason, compromised adult neurogenesis is considered to be part of the cognitive degeneration that characterizes neurological disorders such as HD (Ransome et al., 2012).

Adult neurogenesis has been observed in several HD preclinical models.The reduced proliferation of neural stem cells (NSCs) results as a common thread among most of the evaluated models.For example, a decreased proliferative potential of NSCs has been specifically observed in the hippocampus of YAC128, R6/1, and R6/2 genetic mouse models of HD, while the global cell proliferation resulted unchanged in the SVZ of the respective control animals.In early-stage transgenic HD rats, the global cell proliferation in the SVZ resulted unaltered.Conversely, in late-stage transgenic HD rats, a marked reduction in cell proliferation was found in the SVZ (Lazic et al., 2006; Kohl et al., 2007; Kandasamy et al., 2010; Simpson et al., 2011).Neurogenesis has been also observed in tissues from post-mortem brains of HD patients where,however, NSC proliferation was found to be unaltered (Curtis et al., 2003;Low et al., 2011; Ernst et al., 2014).Interestingly, the decreased proliferation of NSCs in HD seems to be counterbalanced by intensified mitotic events of neuroblasts in the SVZ of several genetic models with different scores of behavioral symptoms (Smith-Geater et al., 2020).A reduction of neurogenesis in the olfactory bulb has been observed in these genetic models, with enhanced migration of neuroblasts towards the lesioned striatum.The atypical behavior of neuroblasts in these models is called “reactive neuroblastosis”.It is characterized by their abnormal proliferation in the SVZ and the migration toward the lesioned striatum is, however, doomed as the cells die before their integration into the circuitry of the striatum (Kandasamy and Aigner, 2018).Notably, this feature has been also observed in postmortem brain samples of HD patients (Curtis et al., 2003; Ernst et al., 2014).

Modulation of neurogenesis in HD

A tight genetic regulation of the adult neurogenesis process can be observed.However, many other players show a role in the control of neurogenesis down-regulating or up-regulating the process.For instance, the up-regulation of neurogenesis occurs within the play of relevant factors such as fibroblast growth factor 2, brain-derived neurotrophic factor (BDNF), antidepressant drugs, estrogens, but also physical exercise, environmental enrichment, and learning.On the other hand, glucocorticoid levels, aging, oxidative stress,and inflammation are all able to counteract adult neurogenesis (Benraiss and Goldman, 2011; Gil-Mohapel et al., 2011).In this view, the possibility of regulation of the adult neurogenic process as a self-regenerative therapeutic strategy can be considered, becoming of the utmost interest in conditions such as HD.For this reason, several attempts have been made to assess whether a positive modulation of neurogenesis can be obtained in preclinical models.In four different R6/1 HD mice models environmental enrichment and physical activity have been tested showing increased BDNF levels, with enhanced survival and cognitive performance as well as reduced levels of intranuclear inclusions in neurons (van Dellen et al., 2000; Spires et al.,2004; Pang et al., 2006; Benn et al., 2010).In another interesting study, the adenovirus-induced overexpression of BDNF and Noggin, as crucial regulators of neurogenesis, in the ventricular wall stimulated the proliferation of striatal newborn neurons in R6/2 HD mice, suggesting an increased neurogenesis.Moreover, the surviving newborn neurons have been found properly integrated in the striatal circuitry as GABAergic neurons, with enhancement of motor function and survival of the mice (Cho et al., 2007).

Focusing on BDNF’s effects on neurogenesis, several studies assessed the possibility of the usage in the HD context of selective serotonin reuptake inhibitors, known to increase BDNF levels in the hippocampus.For instance, in two different studies, the daily administration of Sertraline on N171-82Q HD transgenic mice and R6/2 HD mice counteracted striatal atrophy increasing the motor scores and enhancing survival.Moreover, the inhibitor improved neurogenesis in the hippocampus and normalized BDNF levels in the cortex of the N171-82Q mice and the striatum of both these rodent models (Duan et al., 2008; Peng et al., 2008).An improvement of hippocampal neurogenesis has also been obtained with the administration of Fluoxetine in an R6/1 HD mice model where, however, there was no effect on the motor function or variation in body weight (Grote et al., 2005).Another strategy regarded the administration of fibroblast growth factor 2, as a positive regulator of neurogenesis in the SVZ, in a R6/2 HD mouse model.Notably, the systemic injection of this factor led to an overall improvement of the HD condition in the animals, with increased neurogenesis.In this model, greater cell proliferation in the SVZ has been observed after fibroblast growth factor 2 administration in the HD mice compared to control animals.Also, a proper striatal integration has been reached by the survived newborn neurons (Jin et al., 2005).

Even if adult neurogenesis is slowly but surely gaining a relevant position among the major actors in HD pathology and therapy, there is a long way to go.The preclinical tests are limited in number and variety of conditions.The knowledge around this crucial process is large but not so deep, and this highlights the need for a different focus on the physiology of adult neurogenesis and its role in HD to develop more reliable strategies with different and new models and conditions.

Cell Therapy for Huntington’s Disease

Over the years the knowledge around stem cells grew larger and larger, letting scientists all over the world define strategies based on the plasticity and the versatility of these cells in a plethora of contexts.Among all the ways of using stem cells, one of the most intriguing is the regenerative approach.In fact, to be able to compensate for lesions caused by injuries or diseases using stem cells optimized to act as therapeutic agents in specific areas of the body is the underpinning idea of therapies based on stem cells (Rosser et al., 2022).Such approaches are of the utmost interest in the context of several diseases, in particular neurodegenerative disorders such as HD where the regeneration of neural tissue with a solid neuronal network and neurotrophic support would be able to counteract neurodegeneration at different levels of neuronal circuitry (Connor, 2018; Figure 1).

Stem cells

Stem cells are undifferentiated cells showing several properties that span from proliferation and self-renewal to the possibility to differentiate into more mature cells of different lineages.Stem cells can be classified, for instance, depending on their ability to differentiate in a certain range of cell types.Hence, it is possible to observe embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and NSCs.ESCs are pluripotent stem cells obtained from the inner cell mass of the blastocyst that can endlessly self-renew and differentiate into all the cell types of the nervous system, making these cells the most affordable source, for instance, of neuronal precursors.However, ESCs show ethical concerns and a higher risk of immune rejection and tumor formation when transplanted into host patients compared to the other types of stem cells.Instead, iPSCs being pluripotent stem cells artificially obtained by non-pluripotent cells provide an “ethically free” cell line that can be used to produce autologous neurons for translational medicine with a reduced rate of immune rejection but with a risk of tumor formation (Tartaglione et al., 2017; Sivandzade and Cucullo,2021).In this view, MSCs are the most reliable ones in the therapeutic context.However, further details regarding MSCs will be discussed in the following subsection.Regarding NSCs, these are multipotent stem cells more committed and specialized than the multipotent MSCs with reduced selfrenewal ability and the possibility to differentiate into cells of the brain tissue,such as neurons, astrocytes, and oligodendrocytes (Tuazon et al., 2019b).NSCs can be obtained from embryonic and fetal brains or brain tissue biopsies and are genetically stable cells with low tumorigenic activity (Ottoboni et al.,2020).However, their application in cell therapy needs to overcome several obstacles, among which the immunological incompatibility in case of allogenic usage, their limited sources and proliferation, and ethical concerns (Tartaglione et al., 2017; Sivandzade and Cucullo, 2021).

MSCs

MSCs are multipotent cells that can be found in adipose tissue, bone marrow, spleen, and umbilical cord, and can be differentiated into several cell types such as cells of muscles, bone, fat, and cartilage (Andrzejewska et al., 2019).Due to their exceptional self-renewal ability without losing the multipotency, MSCs show great promise in cell therapy applied to neurodegenerative diseases.In fact, MSCs are easier to collect compared to ESCs with minor ethical concerns and reduced tumorigenesis and immune rejections.Moreover, MSCs can cross the blood-brain barrier via paracellular pathways.From the therapeutic point of view, MSCs can be transplanted through intrathecal or intracerebral injections (Tuazon et al., 2019a).After the delivery, these cells and their secretome stimulate many activities including neurogenesis, microglia activation, anti-inflammatory activity, production of trophic factors like BDNF, and reduction of oxidative stress and apoptosis(Sivandzade and Cucullo, 2021; Kerkis et al., 2022).Among all the types,MSCs showed the most encouraging and potentially translatable results thanks to the synaptogenic, neurogenic, and neuroprotective properties described above, but also thanks to their reduced malignant transformation(Kim et al., 2021).In a rodent model of HD, rat bone marrow-derived MSCs(passage not specified) secreted protective factors such as BDNF, collagen type 1, and fibronectin showing neuroprotective effects and improving behavioral features (Rossignol et al., 2011).In another preclinical study, mice bone marrow-derived MSCs (passage 12) optimized to overexpress nerve growth factor (NGF) and BDNF and transplanted in a YAC 128 transgenic mouse model of HD ameliorated behavioral impairment and striatal neuronal loss, suggesting the role of the overexpressed trophic factors in positively regulating the microenvironment in lesioned brain areas (Dey et al., 2010).Furthermore, human bone marrow-derived MSCs (passages 3 to 5) implanted into the dentate gyrus of the hippocampus of a transgenic mouse model of HD improved neurogenesis and differentiation of endogenous NSCs (Snyder et al., 2010).Similar results have been obtained in another preclinical study where human bone marrow-derived MSCs (passage not specified)transplanted in a rodent model of HD exerted neurotrophic, antiapoptotic,and neuroprotective effects, leading to amelioration of the motor function in the treated animals (Lin et al., 2011).

Therapeutic strategy

The aim of cell therapy approaches is to convey specific cell types in specific brain areas, and, in the case of neurodegenerative disorders, to restore a neural network in lesioned sites.Moreover, the possibility to enhance a trophic auxiliary microenvironment with anin situproduction or delivery of neurotrophic compounds, such as BDNF, vascular endothelial growth factor,or glial-derived neurotrophic factor to assist the neurons in their physiology and counteracting pathology can be a supportive method to reach the cell therapy intent (Tian et al., 2021; Xiao et al., 2022; Choi et al., 2023; Li et al.,2023).In HD, cell therapy approaches have shown to be promising therapeutic strategies in preclinical and clinical studies.The latter will be discussed in detail later in the text (Tartaglione et al., 2017; Bachoud-Lévi et al., 2021).

Knowing that MSNs greatly suffer from the pathogenic mechanisms of HD,among all the neurons, a proposed approach based on the replacement of these neurons with cells capable of differentiation into MSNs has been first tested.The concept behind this approach relies on the observed impossibility to survive of mature neurons when transplanted.Thus, several protocols have been produced to derive MSN-like neurons from ESCs, iPSCs, and NSCs.The application of these cells in HD preclinical studies showed improvements determined by the ability of the grafted cells to differentiate in neurons and glial cells but also to secrete BDNF and other trophic factors, and to properly link to the local endogenous circuitry (Tartaglione et al., 2017).For instance, the cell replacement approach in HD has been investigated using NSCs obtained from iPSCs.In a preclinical study, iPSCs obtained from an HD patient and differentiated into NSCs showed functional and behavioral recovery after 12 weeks from the striatal transplantation in a rat model of HD, with the formation of functional GABAergic neurons, as indicated by electrophysiological recordings.However, clear signs of HD pathology have been observed in the rats after 33 weeks from the graft, highlighting the important role of a long-term follow-up inin vivostudies (Jeon et al., 2012).In a second study, neural precursor cells derived from human iPSCs and transplanted in a mouse model of HD partially replaced the missing neurons,as observed with retrograde axon tracing, enhancing neurogenesis, and exerting anti-inflammatory effects.Also, the treated mice showed behavioral and motor improvements (Yoon et al., 2020).In order to increase the lifespan of the transplanted cells an interesting approach that aims to co-transplant different kinds of stem cells at once has been proposed.Based on the utter relevance of the establishment of a trophic local microenvironment to support the transplanted cells in their integration and differentiation, such an approach combines cells that easily differentiate in neuronal lineages and other stem cells able to prepare the ground with, for instance, the secretion of trophic factors.An example of this can be found in a preclinical study in which adult NSCs and rat bone marrow-derived MSCs (passage 4) have been transplanted alone or in combination in a rat model of HD (Rossignol et al., 2014).The cotransplantation led to increased survival of NSCs in the treated rats compared to the rats treated only with NSCs, probably thanks to the properties of MSCs and their secretome.Moreover, the co-transplantation extended the lifespan of the animals treated compared to control animals and to animals treated with a single type of cells, improving behavioral and motor features.However,even if it can be strongly reduced the risk of immune rejection is present when using stem cells.Also, cellular overgrowth and tumorigenesis should always be considered when using these kinds of stem cells.

Regarding immune rejections, the possibility to provide autologous cells from iPSCs of patients appears as a reliable alternative therapeutic route in the HD context.Applied to HD patients, the concept of deriving autologous cells from their iPSCs relies on the availability of genetic tools to correct the mutation carried by the patients’ cells.Many preclinical studies showed that this is actually a viable way (Bachoud-Lévi et al., 2021).For instance, an attempt at modification via homologous recombination of the CAG mutation carried by HD patient-derived iPSCs has been performed to obtain autologous human NSCs without pathogenic CAG repeats.Interestingly, after 2 weeks the transplantation of the modified cells in the R6/2 HD mice brains led to the formation of MSNs and integration of the novel neurons in the circuitry, with reduced cell death and enhanced bioenergetic impairment (An et al., 2012).

A recent study showed neural progenitor cells derived from iPSCs of an HD rhesus monkey transplanted in the transgenic animal after genetic modification of the expression levels of mHTT by a small-hairpin RNA.The transplantation of the genetically modified cells ameliorated motor function and improved lifespan in HD monkeys compared to the controls, with the integration of the new cells in the local circuitry (Cho et al., 2019).In a more recent study, the genetic editing of autologous neural progenitor cells derived from iPSCs of a transgenic mouse model of HD led to the ablation of the gene SUPT4H1 involved in the transcription of long trinucleotide repeats.The transplant of the modified cells improved the motor function in the treated mice compared to the controls.Moreover, the authors observed the increment of neuronal differentiation of the edited cells compared to the notedited cells, with reduced mHTT expression (Park et al., 2022).The results obtained so far testify the reliability of the autologous transplant approach.However, the feasibility of this strategy for a widespread application is low due to several aspects, among which the costs regarding the production of cells and the genetic modifications, and the regulatory investigations to assess the potential clinical use (Bachoud-Lévi et al., 2021).

Huntington’s disease from a clinical point of view

Several clinical studies have been performed since the 1990s to assess the feasibility of regenerative approaches in HD humans.A first attempt described the bilateral transplantations of fetal cells in putamen and caudate nucleus in a small cohort of HD patients, showing at one year from the surgery no significant side effects from the procedure or the immunosuppressant drugs.Interestingly, the grafts survived and differentiated, as described by resonance imaging, leading to improvement of the motor condition of the patients and proving the feasibility of the transplantation of fetal stem cells in HD patients (Philpottet al., 1997; Kopyov et al., 1998).However, ethical concerns rose regarding the tissue source.Also, the histological autoptic analyses on patients’ brains highlighted a limited neural integration of the grafts in the local circuitry (Freeman et al., 2000).A similar result has been observed, also, in other clinical trials in 2007 and 2009 (Keene et al., 2007,2009).Other attempts contemplate the grafting of ESCs or fetal tissue.In the first case, five patients underwent bilateral striatal grafting one time per year, for 2 years.The results obtained were contradictory as three patients experienced cognitive and motor improvement while the other two patients showed no positive results at all.However, stability and remission have been found in all the patients after 2 years in a 6-year follow-up (Bachoud-Lévi et al., 2000, 2006).In the second trial, the unilateral graft in the striatum showed no adverse effect, demonstrating again the feasibility of the fetal graftin HD humans (Rosser et al., 2002).In a randomized multicenter trial called multicentric intracerebral grafting in HD, the human fetal ganglionic eminence was intracerebrally grafted into 45 HD subjects but no clinical benefits have been observed, probably due to graft rejection (Bachoud-Lévi and on behalf the Multicentric Intracerebral Grafting in Huntington’s Disease Group,2020).As a response to multicentric intracerebral grafting in HD results, a new trial (TRIDENT) started in 2021 with the aim to evaluate in HD patients the safety and viability of incrementing the number of fetal striatal cells intracranially grafted (Drew et al., 2021).Currently, other three clinical trials are ongoing: SAVE-DH, ADORE-DH, and ADORE-EXT.The first one, SAVE-DH,is a phase 1 trial that tries to observe variations in the motor and behavioral functions after intravenous injections of low and high doses of dental pulpderived MSCs (Cellavita HD), assessing the safety of this cell therapy (Azidus,2022c).ADORE-HD is a randomized phase 2 trial in which the effectiveness of Cellavita HD cell therapy will be tested after intravenous injections of high and low doses of dental pulp-derived MSCs (Azidus, 2022b).Finally, ADOREEXT provides an extension to ADORE-HD in order to evaluate higher doses of Cellavita HD (Azidus, 2022a).

Therefore, it is possible to say that cell therapy, as a promising approach to HD, is away from a reliable clinical setting.Nonetheless, cell therapy can count on a large variety of preclinical and clinical studies that let to collect many different data regarding HD models, cell management protocols, welldesigned follow-up, and proper characterizationin vitroandin vivoof the grafts and their effects (Rosser et al., 2022).Now, more than ever, there is the urge to convey all the paths explored into one safe, practicable, and reliable road.

Astrocyte Reprogramming in Huntington’s Disease

Of all the components of the mammalian nervous system glial cells are among the most abundant.Glial cells are non-neuronal cells that can be divided into four major types such as astrocytes, oligodendrocytes, microglia,and ependymal cells, each of which affects the surrounding neurons in different manners.Glial cells play an underpinning physiological regulatory activity in the nervous system (Quan et al., 2022).In fact, glial cells participate in synapse and axon formation, regulate cell metabolism and blood-brain barrier formation, and are involved in cognitive activities such as memory and learning, constantly supporting the homeostasis of the surrounding cell populations (Fields et al., 2014).Moreover, glial cells are involved in the cleaning system removing dead neurons and fighting pathogens (Clarke and Barres, 2013).Among all the glial cells, astrocytes raised the attention of scientists for various aspects.Astrocytes are the most large, abundant, and widespread glial cells characterized by a long list of relevant functions.For instance, astrocytes have been reported to be able to self-renew similarly to stem cells to produce more immature progenitors and neuroblasts (Doetsch et al., 1999; Goldman, 2003).Starting from this observation, scientists tried to take control of this process assessing bothin vitroandin vivothe possibility of reprogramming astrocytes into neurons via activated transcription factors and drugs, as a novel regenerative approach in the context of neurodegenerative disorders such as HD.In this way, starting from astrocytes it is possible to obtain pluripotent cells that can be differentiated into specific different cells but also it is possible to transdifferentiate astrocytes into other target cells avoiding the pluripotency step (Li and Chen, 2016; Chen et al., 2020; Tai et al., 2020).The regenerative approach considers several transcription factors known to be related toin vivoastrocyte reprogramming such as NeuroD1,Neurog2, Ascl1, SOX10, Sox2, NURR1, and shPTB (Niu et al., 2015; Talifu et al.,2023).Lentivirus and adeno-associated viruses (AAVs), chosen as vectors, are micro-injected in the specific site of interest inducing the transformation of a certain percentage of astrocytes (Verdera et al., 2020; Muhuri et al., 2021).In HD, an attempt to reprogram astrocytes into GABAergic neurons has been made.The experiment has been carried out using AAVs with the instructions to obtain the striatal overexpression of NeuroD1 and Dlx2, the second one specific for the GABAergic phenotype, in a mouse model of HD.The transformed astrocytes showed electrophysiological properties like GABAergic neurons, producing also improvements in lifespan and dysfunction of the treated animals (Wu et al., 2020).

Astrocytes and neurons originate from the same progenitors and astrocyte reprogramming can be a feasible technique to avoid, for example, immune rejections (Wan and Ding, 2023).Moreover, the widespread distribution of glial cells into the central nervous system suggests the possibility of intervention throughout the system to support neurons in lesioned sites,highlighting the potential therapeutic role of the astrocytes reprogramming into HD and other neurodegenerative disorders.Even if the interest of the researchers is growing day by day toward astrocytes and their role in regenerative medicine, so much needs to be assessed to think about the translation of this approach from preclinical to clinical context.For instance, a strong preclinical base is needed.

Exosomes and Huntington’s Disease

The literature of the last decades saw as a resolutive strategy the replacement of dead cells with therapeutic cells in lesioned tissues, assuming that the beneficial effects observed in several lesioned models were exerted only by the presence of the new cells.It is now clear that the main actor is the secretome of these cells that play a role in lesioned tissues (Zhang and Cheng,2023).The secretome is characterized by a variety of factors, among which a vesicular component can be observed.The vesicular component comprises extracellular vesicles, particles enveloped in a bilayer of phospholipids physiologically produced by all the cells and that can be classified according to biogenesis, size, and content in microvesicles, apoptotic bodies, and exosomes(Kerkis et al., 2022; Vatsa et al., 2022).Of all the vesicles, exosomes showed peculiar characteristics that led scientists to deepen their knowledge of their composition and function.Interestingly, the involvement of exosomes in cellto-cell interactions has been observed (Huo et al., 2021).Exosomes play a role in the physiological and pathological regulation of intercellular signaling mainly due to their content which can comprise metabolites, trophic factors,but also protein, nucleic acids, and lipids (Kalluri and LeBleu, 2020).Moreover,these vesicles show the ability to easily spread among tissues, even reaching the central nervous system thanks to their nanometric size that spans from 30 nm to 200 nm (Araldi et al., 2020; Xia et al., 2022).Exosomes originate from invaginations of the multivesicular bodies.During this process, small amounts of cytoplasm are enclosed in the vesicles.Due to this, inside exosomes cellspecific cytoplasmic components can be found.When the MVB fuses itself with the plasma membrane the exosomes are secreted in the extracellular environment where are finally free to spread.Here, they can exert their biological activity in several ways.Exosomes, in fact, can directly fuse with the membrane of a target cell or be internalized by the target cell in order to activate or modify signaling events (Gurung et al., 2021).Notably, exosomes can release their contents into the targeted cell cytoplasm during their fusion with the plasma membrane or can maintain their vesicular state when they are internalized.In the latter case, exosomes are collected into MVBs with the possibility of lysosome-mediated degradation or being released again toward near cells (Zhang et al., 2019).Among all their biological tasks, exosomes are known to be involved in several neuronal mechanisms.In fact, these vesicles showed the ability to physiologically modulate synaptic plasticity and activity,neurogenesis, myelination, and neuronal homeostasis restoration after a lesion occurred due to disease or injury (Sharma et al., 2019).However, as in physiology exosomes play a role also in the pathology.For instance, in HD exosomes are known to be responsible for the spreading of mHttprotein as well as for serving as a vehicle for the mHttmRNA.As the invagination on the MVB occurs, cytoplasmic mHttprotein and RNA can often be engulfed inside the vesicles, and through exosomes, they can easily spread out to other cells and brain areas promoting the pathophysiological events that characterize HD (Ananbeh et al., 2021).In the context of the brain lesion caused by HD,the physiological involvement of Htt in vesicular trafficking regulating via phosphorylation both anterograde and retrograde transportation highlighted a fundamental pathological feature of this neurological disorder.In fact, in HD the mHtthas been found to be able to totally derange this process due to its interaction with dynactin and Huntingtin-associated protein 1 involved in the microtubular transportation causing the cessation of the vesicular trafficking of molecules such as BDNF and micro RNAs (miRNAs) (Gauthier et al., 2004;Pardo et al., 2010; Roux et al., 2012).The latter, as regulatory molecules physiologically involved in the suppression of mRNA translation, have been found strategically conveyed by exosomes to exert their regulatory activity in different places.In HD miRNA’s activity resulted altered.For instance, miR-124 which normally reduces the levels of the RE-1 silencing transcription factor (REST) gene involved in the production of BDNF is down-expressed in HD, causing increased levels of REST and BDNF repression (Tung et al., 2021).Thus, knowing the role of miR-124 against BDNF and their involvement,among all, in adult neurogenesis in SVZ a neural regenerative therapeutic strategy aiming to deliver miR-124 into the lesioned area has been proposed.However, due to the instability of miRNA molecules alone, other strategies have been considered.So, given the properties of exosomes and their ability to cross the blood-brain barrier, these nanosized vesicles have been suggested as valuable carriers of small therapeutic molecules such as miRNAs in the central nervous system (Li et al., 2018; Herrmann et al., 2021).Hence, in a preclinical study, the striatal administration of exosomes obtained from HEK-293 cells engineered to over-express miR-124 in an R6/2 HD mouse model led to increased levels of miR-124 compared to controls.The mice showed decreased levels of REST protein in contrast to control animals.Although exosome administration effectively produced specific desired effects in the treated mice, no significant results have been observed in the study, probably due to exosomes’ low levels of miR-124 (Lee et al., 2017).Nevertheless, the tested approach validated the proposed exosome-based regenerative strategy in the HD context.Other attempts have been made to better assess the usefulness of exosomes in molecule delivery.For instance, in the N171-82Q and BACHD mice model of HD the intravenous administration of exosomes loaded with small interfering RNAs (siRNAs), which can selectively silence long-term gene expression by binding specific mRNA, targeting HTT exon 1 transcript, efficiently reduced mHttexpression in mouse brains of 54% and 46% respectively (Wu et al., 2018).Similar effects have been obtained in other studies where siRNAs have been chemically stabilized to increment their loading into exosomes.The loaded exosomes were then tested upon cultured primary cortical neurons and mice’s brains, where a significant reduction of mHtt expression was observed (Didiot et al., 2016).However,in a preclinical study, exosomes obtained from adipose-derived stem cells and administrated in neuronal cells isolated from R6/2 mice exerted beneficial effects, decreasing, for instance, mHtt aggregates and increasing peroxisome proliferator-activated receptor gamma coactivator 1-alpha and phosphorylated cyclic AMP response element-binding protein levels, which are involved in a neuroprotective cascade (Lee et al., 2016).The positive results obtained in the study take into account not only exosome properties but also the relevance of the cell by which exosomes originate.In fact, the choice of MSCs is utterly important due to the collection of molecules and compounds that characterize their cytosol, leading to exosomes charged with trophic molecules as well as with miRNAs, such as miR-214, miR-150, miR-9, miR-22 and many others (Ghose et al., 2011; Jovicic et al., 2013; Ananbeh et al., 2021) that can be all considered in novel therapeutic strategies in HD context and with exosomes as vectors (Figure 2).

Discussion and Future Perspectives

HD is a devastating neurological condition characterized, for instance, by remarked neuronal loss.GABAergic MSNs are the most susceptible among all the involved cells to the catastrophic effects of mHtt.To date, various molecular and genetic approaches are being tested in preclinical and clinical studies with the aim to lower mHtt’s levels or modify aspects of the disease.However, these approaches do not imply the regeneration of the damaged tissues.Thus, regenerative medicine is of the utmost interest in the HD context.For instance, cell therapy showed promising results in HD preclinical and clinical studies.As reported above, several attempts have been made to counteract the neuronal loss caused by HD, obtaining remarkable trophic effects with various kinds of cells.Nonetheless, there are limits to their application.Firstly, the lack of proper amounts of therapeutic cells due to their scarce availability.In many cases, these cells cannot also be stored for an ideal time, making it hard to control their quality and to fit their use with the surgical schedule.Secondly, the lack of standardized surgical procedures,the choice of the proper administration routes, and the ethical and practical concerns related to the number of patients.Another concern regards the availability of cell lines obtained with good manufacturing practices that are often related to higher variability among studies.To date, cell therapy remains the major regenerative approach that aims to structurally and functionally restore lesioned brains.Such an approach must be considered in light of neurogenesis.In fact, cell therapy protocols can comprise the cells to replace the lost ones and to support the lesioned sites with a plethora of trophic factors able to enhance neurogenesis.Still, so much needs to be uncovered to gain proper control of these mechanisms.However, to bypass cell therapy limits, at least regarding the invasiveness of these procedures,other strategies can be used to support lesioned, sites stimulating and strengthening neurogenesis.For instance, thein situreprogramming of astrocytes into neurons is a promising approach that exploits not only the

Figure 2 ｜Wild-type and engineered exosomes exerting trophic effects in HD brain.Exosomes and engineered exosomes are a valuable therapeutic approach for HD due to their content, including neurotrophic factors and protective miRNAs.Created with Adobe Photoshop.BDNF: Brain-derived neurotrophic factor; HD: Huntington’s disease; HTT:huntingtin gene; NGF: nerve growth factor.

availability of a large number of cells ready to be reprogrammed but also the possibility of supporting the lesioned sites without risks such as immune rejections.Another relevant procedure sees the usage of the vesicular content of therapeutic cells, in particular exosomes.Exosomes are considered therapeutic vesicles that can be implied in non-invasive strategies.Thanks to the described properties, exosomes may play a fundamental role in the neural regeneration context.As reported above, exosomes showed trophic effects upon several HD models.Their trophic function has been observed both when exosomes have been used after their isolation from cells that contain cytoplasmic trophic factors and when exosomes have been loaded with therapeutic molecules such as, for example, BDNF or other chemical compounds.

In conclusion, it is possible to affirm that so much has been done to define strategies to counteract the terrible effects of HD upon cells and tissues in order to find a reliable regenerative approach.So far, preclinical and clinical studies have widely assessed the strengths and weaknesses of many different methods establishing the feasibility of neural regenerative therapy in the HD context.Moreover, the recent literature highlighted that adult neurogenesis plays a role both in physiology and pathology, suggesting the therapeutic potential of this dynamic and underpinning process.However, even if the knowledge about neural regeneration and HD has grown bigger and bigger a new focus is needed with the aim to overcome actual and future challenges:an interdisciplinary build-up of strategies may be a resolutive method to reach the goal.

Author contributions:Manuscript conception and design: Md’A, VC; data collection, analysis and interpretation: FD; manuscript writing: FD, VC, and GL;figure preparation: FA; manuscript revision: AC and Md’A; approval of the final version of the manuscript: all authors.

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

Data availability statement:The data are available from the corresponding author on reasonable request.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Julien Rossignol, Cardiff University, UK.

Additional file:Open peer review report 1.