Krabbe’s disease (KD) is caused by mutations in the lysosomal enzyme galactocerebrosidase and is associated with psychosine toxicity. The sphingosine 1-phosphate receptor (S1PR) agonist fingolimod (FTY720) attenuates psychosine-induced cell death of human astrocytes, demyelination in cerebellar slices, as well as demyelination in the central nervous system of twitcher mice. Psychosine also accumulates in the peripheral nervous system in twitcher mice; however, effects of fingolimod on this peripheral myelin have not been examined. The aim of this study was to investigate the effects of fingolimod administration on peripheral and central markers of myelination. Here, we report that fingolimod administration (1 mg/kg/day) from postnatal day 5 (PND) onwards did not alter peripheral demyelination in the sciatic nerve of twitcher mice, despite significantly reducing myelin debris, glial reactivity, and neuronal damage in the cerebellum. We also find fingolimod administration improves twitching and mobility scores in twitcher mice. Importantly, we find that fingolimod significantly increases the lifespan of twitcher mice by approximately 5 days. These findings suggest differential effects of fingolimod on peripheral and central neuropathy in twitcher mice, which may explain its modest efficacy on behavior and lifespan.

Fingolimod hydrochloride (FH) has emerged as a vital medication for managing Multiple Sclerosis (MS). Despite its high oral bioavailability of 93

Krabbe disease (KD) is a rare demyelinating disorder characterized by demyelination caused by mutations in the GALC gene, resulting in toxic accumulation of psychosine. Psychosine has been identified as detrimental to oligodendrocytes, leading to demyelination through diverse hypothesized pathways. Reducing demyelination is essential to maintain neurological function in KD; however, therapeutic interventions are currently limited. Acetylcholinesterase inhibitors (AChEi) are commonly used for symptomatic management of Alzheimer's Disease and are suggested to have potential disease-modifying effects, including regulating myelin state. In particular, donepezil, an AChEi, has demonstrated promising effects in cellular and animal models, including promotion of the expression of myelin-related genes and reduction of glial cell reactivity. This drug also acts as an agonist for sigma-1 receptors (Sig-1R), which are implicated in demyelination diseases. In the context of drug repurposing, here, we demonstrate that administration of donepezil has protective effects in the twitcher mouse model of KD. We provide data showing that donepezil preserves myelin and reduces glial cell reactivity in the brains of twitcher mice. Moreover, donepezil also improves behavioral phenotypes and increases lifespan in twitcher animals. These findings suggest that donepezil, with its dual activity as an AChE inhibitor and Sig-1R agonist, may hold promise as a therapeutic candidate for demyelinating diseases, including KD.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of the arylsulfatase A lysosomal enzyme and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that sulfatide induces demyelination in a concentration-dependent manner. Interestingly, our novel data demonstrate that sulfatide-induced demyelination is underpinned by PARP-1 activation, oligodendrocyte loss, pro-inflammatory cytokine expression, astrogliosis, and microgliosis. Moreover, such sulfatide-induced effects can be attenuated by the treatment with the poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor Olaparib (IC50∼100 nM) suggesting that this small molecule may be neuroprotective and limit toxin-induced demyelination. Our data support the idea that sulfatide is a key driver of demyelination and neuroinflammation in MLD and suggest that PARP-1 inhibitors have therapeutic utility in the sphere of rare demyelinating disease.

The role of altered myelin in the onset and development of schizophrenia and changes in myelin due to antipsychotics remains unclear. Antipsychotics are D-2 receptor antagonists, yet D-2 receptor agonists increase oligodendrocyte progenitor numbers and limit oligodendrocyte injury. Conflicting studies suggest these drugs promote the differentiation of neural progenitors to oligodendrocyte lineage, while others report antipsychotics inhibit the proliferation and differentiation of oligodendrocyte precursors. Here, we utilised in-vitro (human astrocytes), ex-vivo (organotypic slice cultures) and in-vivo (twitcher mouse model) experimental study designs of psychosine-induced demyelination, a toxin that accumulates in Krabbe disease (KD), to investigate direct effects of antipsychotics on glial cell dysfunction and demyelination. Typical and atypical antipsychotics, and selective D-2 and 5HT(2A) receptor antagonists, attenuated psychosine-induced cell viability, toxicity, and morphological aberrations in human astrocyte cultures. Haloperidol and clozapine reduced psychosine-induced demyelination in mouse organotypic cerebellar slices. These drugs also attenuated the effects of psychosine on astrocytes and microglia and restored non-phosphorylated neurofilament levels, indicating neuroprotective effects. In the demyelinating twitcher mouse model of KD, haloperidol improved mobility and significantly increased the survival of these animals. Overall, this study suggests that antipsychotics directly regulate glial cell dysfunction and exert a protective effect on myelin loss. This work also points toward the potential use of these pharmacological agents in KD.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5–100 μM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca 2+ ) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC 50 ∼20 μM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC 50 ∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca 2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD. Graphical Abstract Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca 2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca 2+ concentration. PARP-1 inhibition with Olaparib reduces Ca 2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy, with dismal survival rates of ~14.6 months. The current standard-of-care consists of surgical resection and chemoradiotherapy, however the treatment response is limited by factors such as tumour heterogeneity, treatment resistance, the blood–brain barrier, and immunosuppression. Several immunotherapies have undergone clinical development for GBM but demonstrated inadequate efficacy, yet future combinatorial approaches are likely to hold more promise. Olaparib is FDA-approved for BRCA-mutated advanced ovarian and breast cancer, and clinical studies have revealed its utility as a safe and efficacious radio- and chemo-sensitiser in GBM. The ability of Olaparib to enhance natural killer (NK) cell-mediated responses has been reported in prostate, breast, and lung cancer. This study examined its potential combination with NK cell therapies in GBM by firstly investigating the susceptibility of the GBM cell line T98G to NK cells and, secondly, examining whether Olaparib can sensitise T98G cells to NK cell-mediated responses. Here, we characterise the NK receptor ligand profile of T98G cells and demonstrate that Olaparib does not dampen T98G susceptibility to NK cells or elicit immunomodulatory effects on the function of NK cells. This study provides novel insights into the potential combination of Olaparib with NK cell therapies for GBM.

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Over the past decade, Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have arisen as a novel and promising targeted therapy for breast cancer gene (BRCA)-mutated ovarian and breast cancer patients. Therapies targeting the enzyme, PARP-1, have since established their place as maintenance drugs for cancer. Here, we present existing evidence that implicates PARP-1 as a player in the development and progression of both malignancy and demyelinating disease. These findings, together with the proven clinical efficacy and marketed success of PARP-1 inhibitors in cancer, present the repurposing of these drugs for demyelinating diseases as a desirable therapeutic concept. Indeed, PARP-1 inhibitors are noted to demonstrate neuroprotective effects in demyelinating disorders such as multiple sclerosis and Parkinson's disease, further supporting the use of these drugs in demyelinating, neuroinflammatory, and neurodegenerative diseases. In this review, we discuss the potential for repurposing PARP-1 inhibitors, with a focus on rare demyelinating diseases. In particular, we address the possible use of PARP-1 inhibitors in examples of rare leukodystrophies, for which there are a paucity of treatment options and an urgent need for novel therapeutic approaches.

Therapeutic strategies for Alzheimer’s disease (AD) have largely focused on the regulation of amyloid pathology while those targeting tau pathology, and inflammatory mechanisms are less explored. In this regard, drugs with multimodal and concurrent targeting of Aβ, tau, and inflammatory processes may offer advantages. Here, we investigate one such candidate drug in the triple transgenic 3xTg-AD mouse model of AD, namely the disease-modifying oral neuroimmunomodulatory therapeutic used in patients with multiple sclerosis, called fingolimod. In this study, administration of fingolimod was initiated after behavioral symptoms are known to emerge, at 6 months of age. Treatment continued to 12 months when behavioral tests were performed and thereafter histological and biochemical analysis was conducted on postmortem tissue. The results demonstrate that fingolimod reverses deficits in spatial working memory at 8 and 12 months of age as measured by novel object location and Morris water maze tests. Inflammation in the brain is alleviated as demonstrated by reduced Iba1-positive and CD3-positive cell number, less ramified microglial morphology, and improved cytokine profile. Finally, treatment with fingolimod was shown to reduce phosphorylated tau and APP levels in the hippocampus and cortex. These results highlight the potential of fingolimod as a multimodal therapeutic for the treatment of AD.

Polymeric nanoparticles are being extensively investigated as an approach for brain delivery of drugs, especially for their controlled release and targeting capacity. Nose-to-brain administration of nanoparticles, bypassing the blood brain barrier, offers a promising strategy to deliver drugs to the central nervous system. Here, we investigated the potential of hybrid nanoparticles as a therapeutic approach for demyelinating diseases, more specifically for Krabbe's disease. This rare leukodystrophy is characterized by the lack of enzyme galactosylceramidase, leading to the accumulation of toxic psychosine in glial cells causing neuroinflammation, extensive demyelination and death. We present evidence that lecithin/chitosan nanoparticles prevent damage associated with psychosine by sequestering the neurotoxic sphingolipid via physicochemical hydrophobic interactions. We showed how nanoparticles prevented the cytotoxicity caused by psychosine in cultured human astrocytes in vitro, and how the nanoparticle size and PDI augmented while the electrostatic charges of the surface decreased, suggesting a direct interaction between psychosine and the nanoparticles. Moreover, we studied the effects of nanoparticles ex vivo using mouse cerebellar organotypic cultures, observing that nanoparticles prevented the demyelination and axonal damage caused by psychosine, as well as a moderate prevention of the astrocytic death. Taken together, these results suggest that lecithin-chitosan nanoparticles are a potential novel delivery system for drugs for certain demyelinating conditions such as Krabbe's disease, due to their dual effect: not only are they an efficient platform for drug delivery, but they exert a protective effect themselves in tampering the levels of psychosine accumulation.

The EBI2 receptor regulates the immune system and is expressed in various immune cells including B and T lymphocytes. It is also expressed in astrocytes in the central nervous system (CNS) where it regulates pro-inflammatory cytokine release, cell migration and protects from chemically induced demyelination. Its signaling and expression are implicated in various diseases including multiple sclerosis, where its expression is increased in infiltrating immune cells in the white matter lesions. Here, for the first time, the EBI2 protein in the CNS cells in the human brain was examined. The function of the receptor in MO3.13 oligodendrocytes, as well as its role in remyelination in organotypic cerebellar slices, were investigated. Human brain sections were co-stained for EBI2 receptor and various markers of CNS-specific cells and the human oligodendrocyte cell line MO3.13 was used to investigate changes in EBI2 expression and cellular migration. Organotypic cerebellar slices prepared from wild-type and cholesterol 25-hydroxylase knock-out mice were used to study remyelination following lysophosphatidylcholine (LPC)-induced demyelination. The data showed that EBI2 receptor is present in OPCs but not in myelinating oligodendrocytes in the human brain and that EBI2 expression is temporarily upregulated in maturing MO3.13 oligodendrocytes. Moreover, we show that migration of MO3.13 cells is directly regulated by EBI2 and that its signaling is necessary for remyelination in cerebellar slices post-LPC-induced demyelination. The work reported here provides new information on the expression and role of EBI2 in oligodendrocytes and myelination and provides new tools for modulation of oligodendrocyte biology and therapeutic approaches for demyelinating diseases.

In the brain, REST (Repressor Element‐1 Silencing Transcription factor) is a key regulator of neuron cell‐specific gene expression. Nuclear translocation of neuronal REST has been shown to be neuroprotective in a healthy ageing context. In contrast, inability to upregulate nuclear REST is thought to leave ageing neurons vulnerable to neurodegenerative stimuli, such as Alzheimer’s disease (AD) pathology. Hippocampal and cortical neurons are known to be particularly susceptible to AD‐associated neurodegeneration. However, REST expression has not been extensively characterised in the healthy ageing brain. Here, we examined the spatiotemporal immunolocalisation of REST in the brains of healthy ageing wild‐type Fischer‐344 and transgenic Alzheimer’s disease rats (TgF344‐AD). Nuclear expression of REST increased from 6 months to 18 months of age in the hippocampus, frontal cortex and subiculum of wild‐type rats, but not in TgF344‐AD rats. No changes in REST were measured in more posterior cortical regions or in the thalamus. Interestingly, levels of the presynaptic marker synaptophysin, a known gene target of REST, were lower in CA1 hippocampal neurons of 18‐month TgF344‐AD rats compared to 18‐month wild‐types, suggesting that elevated nuclear REST may protect against synapse loss in the CA1 of 18‐month wild‐type rats. High REST expression in ageing wild‐type rats did not, however, protect against axonal loss nor against astroglial reactivity in the hippocampus. Taken together, our data confirm that changes in nuclear REST expression are context‐, age‐ and brain region‐specific. Moreover, key brain structures involved in learning and memory display elevated REST expression in healthy ageing wild‐type rats but not TgF344‐AD rats.

Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination bothin vitroandex vivomodels. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. Thesein vivofindings corroborate initialin vitrostudies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease.SIGNIFICANCE STATEMENTThis study demonstrates that the administration of the therapy known as fingolimod in a mouse model of Krabbe's disease (namely, the twitcher mouse model) significantly rescues myelin levels. Further, the drug fingolimod also regulates the reactivity of glial cells, astrocytes and microglia, in this mouse model. These protective effects of fingolimod result in an increased life span of twitcher mice.

Piezo1 is a mechanosensitive ion channel that facilitates the translation of extracellular mechanical cues to intracellular molecular signaling cascades through a process termed, mechanotransduction. In the central nervous system (CNS), mechanically gated ion channels are important regulators of neurodevelopmental processes such as axon guidance, neural stem cell differentiation, and myelination of axons by oligodendrocytes. Here, we present evidence that pharmacologically mediated overactivation of Piezo1 channels negatively regulates CNS myelination. Moreover, we found that the peptide GsMTx4, an antagonist of mechanosensitive cation channels such as Piezo1, is neuroprotective and prevents chemically induced demyelination. In contrast, the positive modulator of Piezo1 channel opening, Yoda-1, induces demyelination and neuronal damage. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that GsMTx4 attenuates demyelination induced by the cytotoxic lipid, psychosine. Importantly, we confirmed the potential therapeutic effects of GsMTx4 peptide in vivo by co-administering it with lysophosphatidylcholine (LPC), via stereotactic injection, into the cerebral cortex of adult mice. GsMTx4 prevented both demyelination and neuronal damage usually caused by the intracortical injection of LPC in vivo; a well-characterized model of focal demyelination. GsMTx4 also attenuated both LPC-induced astrocyte toxicity and microglial reactivity within the lesion core. Overall, our data suggest that pharmacological activation of Piezo1 channels induces demyelination and that inhibition of mechanosensitive channels, using GsMTx4, may alleviate the secondary progressive neurodegeneration often present in the latter stages of demyelinating diseases.

Astrocytes are important for information processing in the brain and they achieve this by fine-tuning neuronal communication via continuous uptake and release of biochemical modulators of neurotransmission and synaptic plasticity. Often overlooked are their important functions in mechanosensation. Indeed, astrocytes can detect pathophysiological changes in the mechanical properties of injured, ageing, or degenerating brain tissue. We have recently shown that astrocytes surrounding mechanically-stiff amyloid plaques upregulate the mechanosensitive ion channel, Piezo1. Moreover, ageing transgenic Alzheimer's rats harboring a chronic peripheral bacterial infection displayed enhanced Piezo1 expression in amyloid plaque-reactive astrocytes of the hippocampus and cerebral cortex. Here, we have shown that the bacterial endotoxin, lipopolysaccharide (LPS), also upregulates Piezo1 in primary mouse cortical astrocyte cultures in vitro. Activation of Piezo1, via the small molecule agonist Yoda1, enhanced Ca2+ influx in both control and LPS-stimulated astrocytes. Moreover, Yoda1 augmented intracellular Ca2+ oscillations but decreased subsequent Ca2+ influx in response to adenosine triphosphate (ATP) stimulation. Neither blocking nor activating Piezo1 affected cell viability. However, LPS-stimulated astrocyte cultures exposed to the Piezo1 activator, Yoda1, migrated significantly slower than reactive astrocytes treated with the mechanosensitive channel-blocking peptide, GsMTx4. Furthermore, our data show that activating Piezo1 channels inhibits the release of cytokines and chemokines, such as IL-1β, TNFα, and fractalkine (CX3 CL1), from LPS-stimulated astrocyte cultures. Taken together, our results suggest that astrocytic Piezo1 upregulation may act to dampen neuroinflammation and could be a useful drug target for neuroinflammatory disorders of the brain.

The group I metabotropic glutamate (mGlu) receptor subtypes, mGlu1 and mGlu5, strongly regulate hippocampal synaptic plasticity. Both harbor PSD-95/discs-large/ZO-1 (PDZ) motifs at their extreme carboxyl terminals, which allow interaction with the PDZ domain of Tamalin, regulate the cell surface expression of group I mGlu receptors, and may modulate their coupling to signaling proteins. We investigated the functional role of this interaction in hippocampal long-term depression (LTD). Acute intracerebral treatment of adult rats with a cell-permeable PDZ-blocking peptide (pep-mGluR-STL), designed to competitively inhibit the interaction between Tamalin and group 1 mGlu receptors, prevented expression of LTD in the hippocampal CA1 region without affecting long-term potentiation (LTP) or basal synaptic transmission. Pep-mGluR-STL prevented facilitation by the group I mGlu receptor agonist, (S)-3,5-Dihydroxyphenylglycine (DHPG), and the mGlu5 agonist, (R,S)-2-chloro-5-Hydroxyphenylglycine (CHPG), of short-term depression (STD) into LTD, suggesting that Tamalin preferentially acts by mediating signaling through mGlu5. These data support that Tamalin is essential for the persistent expression of LTD and that it subserves the effective signaling of group 1 mGlu receptors.