Neurotherapeutics

Parkinsons disease (PD) affects millions worldwide, with no efficient therapy currently available. A major cause of the initiation and progression of this degenerative disease is the dysfunctional cellular protein quality control system (PQC), leading to the accumulation of toxic protein aggregates in neurons. We previously reported azadiradione (AZD), a small molecule (MW 451 Da), as a potent inducer of heat shock factor 1 (HSF1) activity, which could alleviate cellular toxicity induced by misfolded proteins by upregulating the levels of inducible molecular chaperones and proteasome activity. Here, we show the multifaceted effect of AZD in enhancing the capacity of PQC machinery in cells, fruit flies, and a PD mouse model. AZD activated HSF1 by promoting its phosphorylation at S326 through MEK. In parallel, AZD boosted protein degradation through increased chymotrypsin-like proteasome activity, upregulation of the ubiquitin ligase CHIP. AZD induced autophagy, marked by elevated levels of Beclin 1, ATG7, and ULK1 phosphorylation at S555, along with mTORC1 inhibition via AMPK activation. Surprisingly, the calorie restriction pathway was also upregulated upon AZD treatment, as demonstrated by the enhanced phosphorylation of FOXO3 and FOXO1, along with increased activity of their target enzymes SOD and catalase. Notably, AKT activity was also suppressed in AZD-treated cells. In vivo, AZD improved motor function, dopaminergic neuron survival, and tyrosine hydroxylase activity in an MPTP-induced mouse model of PD, and extended lifespan in Drosophila without compromising fertility or mobility. These findings highlight AZD as a promising therapeutic candidate for restoring PQC and mitigating PD pathology.

In this randomized, double-blind, controlled trial of 8 weeks of repetitive transcranial magnetic stimulation (rTMS) for chronic pain, we compared the classic primary motor cortex (M1) rTMS with a novel target-selection strategy based on pre-therapy cortical connectivity. Guided by principles of homeostatic plasticity, we tested whether stimulating the cortical site with the lowest pre-therapy global connectivity would be more effective than two active comparators: stimulating the site with the highest pre-therapy global connectivity or stimulating M1 independent of connectivity. Before starting rTMS treatment, TMS-evoked EEG potentials were recorded from four cortical targets: M1, the dorsolateral prefrontal cortex, the anterior cingulate cortex, and the posterosuperior insular cortex. For each target, global connectivity was quantified using a distance-weighted, phase-based index (debiased weighted phase lag index, wPLI) derived from pre- and post-TMS-evoked EEG activity, capturing both the magnitude and spatial extent of TMS-induced oscillatory phase locking across cortical regions. Target allocation in the Low- and High-Connectivity groups was based on this global connectivity measure. Ninety patients with chronic pain were randomized to Low-Connectivity, High-Connectivity, or Classic-M1 groups. Treatment consisted of 12 rTMS sessions delivered over 8 weeks to the assigned target. The primary outcome was the proportion of patients achieving [≥] 30% reduction in pain intensity. Secondary outcomes included continuous change in pain intensity, pain interference, sleep, fatigue, mood, quality of life, and patient global impression of change. No between-group differences were observed for primary or secondary outcomes (p > 0.05). In prespecified exploratory analyses, we examined whether pre-therapy local connectivity (within-target wPLI) predicted treatment response. In the Classic-M1 group, lower pre-therapy local M1 connectivity was associated with a greater reduction in pain intensity (r = 0.50, p = 0.005). This association was not observed in the Low- or High-Connectivity groups. A regression model including group-by-connectivity interaction indicated that the relationship between local connectivity and pain reduction differed between the Classic-M1 and High-Connectivity groups (p = 0.038). The results of this clinical trial showed that connectivity-based target allocation using global connectivity did not improve clinical outcomes. However, lower local M1 connectivity was associated with greater pain reduction following Classic-M1 stimulation, suggesting that local M1 connectivity may serve as a potential biomarker of response.

Psychedelics are well known for their ability to produce profoundly altered states of consciousness. But, more importantly, the effects of psychedelics can influence neurobehavioral changes that last well after these acute subjective effects end. This phenomenon is currently being leveraged in the development of psychedelic-assisted psychotherapies for the treatment of multiple neuropsychiatric disorders. The cellular and molecular mechanisms by which single doses of psychedelics are able to mediate long-term cognitive changes are an active area of research. We hypothesize that psychedelics contribute to long term changes in cellular state by covalently modifying proteins. This post-translational modification by psychedelics is possible through the transglutaminase-mediated transamidation of their amine termini to glutamine carboxamide residues. Here, we synthesize and utilize a propargylated analogue of mescaline - the classic serotonergic psychedelic phenethylamine found in cacti species - to identify putative protein targets of psychedelic modifications through the use of click-chemistry in a primary human astrocyte cell culture model. Our preliminary findings indicate that a diverse array of glial proteins may be substrates for transglutaminase 2-mediated monoaminylation by our model phenethylamine ("phenethylaminylation"). Based on these points, we speculatively highlight new directions for the study of this putative noncanonical psychedelic activity.

Chronic Primary Pain (CPP) is a new diagnostic category including chronic pain conditions lacking clinical signs or a clear etiopathogenetic origin. These disorders may share a common neural mechanism known as central sensitization, where nociceptive neurons become hyper-responsive to standard or subthreshold pain stimuli, resulting in pain hypersensitivity. In this context, non-invasive brain stimulation (NIBS) seems a promising tool to improve CPP symptoms by targeting maladaptive brain activity and connectivity. To date, NIBS effects on CPP symptoms remain unexplored. To fill this gap, we conducted a meta-analysis, investigating the effect of NIBS in improving the three core symptoms of CPP, namely pain intensity, emotional distress, and functional disability. Following PRISMA guidelines, we screened four databases up to the end of January 2023. Thirty-five English-written randomized clinical trials were included, comprising 874 participants assigned to the real stimulation condition and 713 to the sham. Findings highlighted the effect of the real over the sham stimulation in improving CPP core symptoms immediately after the treatment. For pain intensity and functional disability, the improvement persisted also at the one-month follow-up. Meta-regression analyses highlighted that a longer CPP duration reduced the effects of NIBS, while an increased number of sessions was associated with greater pain relief at follow-up. Taken together, our results suggest that NIBS can effectively alleviate CPP symptoms in the short and medium term. Further research is needed to define standardized NIBS protocols for CPP management and explore whether combining NIBS with other therapeutic interventions can enhance effects duration and efficacy.

Melatonin has emerged as a promising pharmacological candidate for bipolar disorder (BD), though its mechanisms of action remain incompletely understood. Its antioxidant, anti-inflammatory, and anti-dopaminergic properties suggest potential relevance to BD pathophysiology. This study investigated melatonins effects on dopamine signalling, metabolism, and oxidative stress under inflammatory and hyperdopaminergic conditions in differentiated SH-SY5Y neuronal cells. Cells were pretreated with 100nM melatonin or vehicle for 2 hours, then exposed to vehicle, IL-6 (20ng/mL), dopamine (5{micro}M or 500{micro}M), or dopamine (500{micro}M) with ascorbic acid (1mM) for 12 or 24 hours. Dopaminergic markers were assessed via real-time PCR and HPLC; metabolic outcomes were measured using Seahorse assay, central carbon metabolomics, in-cell Western assay, and glucose uptake assay; and oxidative stress was evaluated via reactive oxygen species (ROS), superoxide (SOX), and total antioxidant capacity (TAC) assays. IL-6 increased dopamine levels, p-Erk1/2/Erk1/2, p-AMPK/AMPK, nucleotide pools, and TAC, while reducing dopamine turnover, SV2C expression, and spare respiratory capacity. Melatonin alone increased nucleotides and NADH, while reducing dopamine turnover, ROS, and glucose-1-phosphate. In IL-6 conditions, melatonin pretreatment enhanced spare respiratory capacity, glucose uptake, and NADH, while reducing dopamine, TAC, p-AMPK/AMPK, p-GSK3{beta}/GSK3{beta}, and non-mitochondrial oxygen consumption. High-dose dopamine (500{micro}M) elevated SOX, p-Erk1/2/Erk1/2, insulin receptor-, GLUT1, glycolytic ATP (glycoATP), and non-mitochondrial oxygen consumption. Melatonin pretreatment attenuated p-Erk1/2/Erk1/2 and GLUT1 elevations. Combined dopamine and ascorbic acid further increased glycolytic intermediates, ROS, p-AMPK/AMPK, and TAC, while reducing p-Erk1/2/Erk1/2, p-mTOR, GLUT1, glucose uptake, and glycoATP. Overall, melatonin mitigated IL-6-induced dopaminergic, oxidative, and metabolic alterations, and partially protected against dopamine-induced metabolic shifts. These findings suggest melatonin may alleviate manic symptoms in BD via both direct dopaminergic modulation and indirect antioxidant and metabolic regulatory effects.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy uses a single, high titre intravenous bolus of AAV9-SMN resulting in impressive, yet limited amelioration of the clinical phenotype. However, risks of this treatment include liver toxicity. Intrathecal administration is under clinical trial but was interrupted due to safety concerns in a concomitant animal study. As there is no direct comparison between the different delivery strategies while avoiding high dose toxicity, we injected SMA mice with low dose scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. Here, IV injections restored SMN in peripheral tissues but not CNS, while ICV injections mildly increased SMN in the periphery and the CNS. Consequently, only ICV treatment rescued motor neuron degeneration. Surprisingly, both treatments resulted in an impressive rescue of survival, weight, motor function, and peripheral phenotypes including liver and pancreas pathology. Our work highlights independent contributions of peripheral organs to SMA pathology and suggests that treatments should not be restricted to the motor neuron. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/467795v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e4e882org.highwire.dtl.DTLVardef@15ccf93org.highwire.dtl.DTLVardef@3fe393org.highwire.dtl.DTLVardef@75cc20_HPS_FORMAT_FIGEXP M_FIG C_FIG

The extent of white matter (WM) and Grey matter (GM) structural neuroplasticity following cognitive behavioral therapy for chronic pain management remains undetermined. In the current study, we investigated structural alterations in GM morphometry, as well as WM complexity and connectivity, before and after an 11-week group CBT for the treatment of chronic musculoskeletal pain. We hypothesized that effective pain management would influence WM structural metrics indicative of brain plasticity, particularly within cognitive and limbic circuitry as well as GM volume within pain matrix structures. To determine this, patients were randomized into two groups: 1) CBT group that received CBT once-weekly for 11-weeks, or 2) EDU group consisting of an active patient control group that received educational materials by mail. All subjects completed behavioral assessments and underwent neuroimaging at: baseline prior to any intervention (TP1), 11-weeks following either CBT or EDU (TP2), and four months following completion of the intervention (TP3). CBT resulted in significant clinical improvements, assessed via behavioral self-reports, compared to EDU. Compared to EDU, region of interest WM analysis revealed several fiber tracts that had significantly increased WM complexity following CBT intervention, including the bilateral posterior internal capsule and the left cingulum within the temporal lobe. Conversely, several tracts exhibited a decrease in WM complexity including the right external capsule, the left posterior internal capsule, and the right cingulum within the temporal lobe. Changes in clinical outcomes were predictive of alterations in WM complexity metrics immediately following intervention and at long-term follow-up. No between-group differences were observed in either WM connectivity or GM volume. In conclusion, psychotherapeutic interventions such as group CBT influence coping strategies for effective pain relief that influence WM microstructure, however, the mechanisms of these changes remain undetermined. Future studies will be required to uncover the biological underpinnings of these alterations in pain populations. ClinicaltrialsgovCan Therapy Alter CNS Processing of Chronic Pain: A Longitudinal Study (https://clinicaltrials.gov/ct2/show/NCT01794988?term=naylor&cntry=US&state=US%3AVT&draw=2&rank=1;NCT01794988). The study protocol was registered in the Clinical Trials Database.

Transcranial direct current stimulation (tDCS) might modulate neural activity and promote neural plasticity. This factorial randomized clinical trial compared a-tDCS on the left dorsolateral prefrontal cortex (l-DLPFC) or sham (s-tDCS), and a-tDCS or s-tDCS on the primary motor cortex (M1) in the connectivity analyses in eight regions of interest (ROIs) across eight resting-state electroencephalography (EEG) frequencies. We included 48 women with fibromyalgia, aged 30 to 65, randomly assigned to 2:1:2:1 to receive 20 sessions during 20 minutes of a-tDCS 2mA or s-tDCS at home, over l-DLPFC or M1, respectively. EEG recordings were obtained before and after treatment with eyes open (EO) and eyes closed (EC). In the EC condition, comparing pre to post-treatment, the a-tDCS on l-DLPFC decreased the lagged coherence connectivity in the delta frequency band between the right insula and left anterior cingulate cortex (ACC) (t=-3.542, p=.048). The l-DLPFC a-tDCS compared to s-tDCS decreased the lagged coherence connectivity in the delta frequency band between the right insula and left ACC (t=-4.000, p=.017). In the EO condition, the l-DLPFC a-tDCS compared to M1 s-tDCS increased the lagged coherence connectivity between the l-DLPFC and left ACC in the theta band (t=-4.059, p=.048). Regression analysis demonstrated that the a-tDCS effect on the l-DLPFC was positively correlated with sleep quality, while a-tDCS on l-DLPFC and M1 s-tDCS were positively correlated with pain catastrophizing. The application of a-tDCS over the l-DLPFC has modulated the connectivity between various brain regions involved in the affective-attentional aspects of pain, especially at lower EEG frequencies during the resting state. These findings suggest that the effects of a-tDCS on neural oscillations could serve as a neural marker associated with its impact on fibromyalgia symptoms.

Despite a wealth of preclinical studies establishing neuroprotective and neurorestorative properties of glial cell-line-derived neurotrophic factor (GDNF) in animal models of Parkinsons disease (PD), clinical trials utilizing direct intracranial infusion of GDNF protein, or adeno-associated virus (AAV)-mediated GDNF gene transfer has not achieved the desired efficacy, largely due to challenges in delivery methods. Given GDNFs strong potential for neuroprotection, alternative strategies to elevate its expression by beyond invasive injection or genetic manipulation remain a promising therapeutic avenue for PD. We previously reported that prokineticin signaling provides a compensatory protective response against dopaminergic neuronal degeneration in cell and animal models of PD. Herein, we report a novel finding that PK2 regulates GDNF gene expression in astrocytes, suggesting that PK2 signaling can be harnessed for neuroprotection in PD. Treatment of cultured astrocytes with the PK2 protein, PK2 gene overexpression or prokineticin receptor 1 (PKR1) agonist IS20 significantly induced the GDNF gene expression and the protein secretion, resulting in enhanced dopaminergic cell survival in cell culture models of PD. Importantly, systemic administration of IS20 through intraperitoneal or intranasal routes elevated GDNF levels in the mouse brain, including the nigrostriatal system. Furthermore, IS20 treatment conferred significant neuroprotective effects in both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced and MitoPark transgenic mouse models of PD. Collectively, our translational findings suggest that pharmacological modulation PK2 signaling may unlock the full clinical benefit of GDNF, offering a novel and non-invasive therapeutic strategy for Parkinsons disease.

Repetitive transcranial magnetic stimulation (rTMS) to the primary motor cortex (M1) provides significant pain relief in [~]45% of chronic pain patients. Identifying biomarkers that predict treatment response before starting rTMS is essential for guiding clinical decision-making. Here, we used TMS combined with electroencephalography (TMS-EEG) to assess pre-treatment cortical function in 43 patients with chronic pain before receiving 12 sessions of therapeutic 10 Hz rTMS to M1 over eight weeks as a secondary analysis from a trial comparing effects of rTMS in different cortical targets. Responders were defined as individuals reporting a [&ge;]30% reduction in pain intensity on a visual analogue scale at week 8. Pre-therapy TMS-evoked cortical reactivity was quantified at M1 using global mean field power (GMFP), and local mean field power (LMFP). Oscillatory dynamics were measures by event-related spectral perturbation (ERSP), and intertrial coherence (ITC) in alpha (8-12 Hz), low-beta (13-20 Hz), and high-beta (21-30 Hz) bands. Compared with non-responders, responders (n=20; 47%) showed lower GMFP, LMFP, alpha-band ERSP, and ITC at the stimulation site (all p<0.05). These low measures correlated with greater reductions in pain intensity (p<0.05). Exploratory supervised machine-learning analysis using three TMS-EEG features (GMFP, alpha-band ERSP, alpha-band ITC) predicted responder status with acceptable performance (ROC-AUC = 0.70, PR-AUC = 0.76). These findings suggest that lower pre-treatment TMS-evoked cortical reactivity and alpha-band oscillatory dynamics may identify patients more likely to benefit from rTMS. Prospective clinical trials should test pre-therapy reactivity and connectivity metrics to select patients more likely to benefit from therapy.

BackgroundChronic neuropathic pain is a complex experience, posing a major challenge in personalizing its treatment. Present treatments consist of non-specific, standardized drugs that are often addictive, leaving many patients non-respondent and with significant side effects. Designing individualized therapies requires targeting the multidimensionality of pain and developing objective endpoints to demonstrate their effectiveness. Currently, non-pharmacological alternatives are emerging, such as neurostimulation and Virtual Reality (VR), activating pain relief via peripheral neuromodulation and attention modulation. Similarly to drugs, many neurostimulation approaches are unspecific, targeting areas near the pain site and disregarding the neural pathway of pain. Above all, neurostimulation and VR are yet to be evaluated as a combined synergistic intervention, particularly in a randomized controlled trial (RCT). Methods and FindingsTo this aim, we developed a targeted neurostimulation congruent with immersive VR platform providing a multisensory pain intervention through the synergistic application of somatotopic electro-tactile and visual stimuli. The endpoints included measuring sensory, neurophysiological (EEG), and self-reported indicators of pain. We tested the efficacy of the multisensory intervention against the control consisting of VR-only intervention on four consecutive intervention days in an RCT (N=18 neuropathic patients). The multisensory intervention resulted in a clinically significant reduction of pain (>50%), lasting up to one-week follow-up. The provided analgesic effect was statistically stronger compared to the VR-only control across treatment days and at follow-up. The clinically relevant pain decrease was accompanied with objective improvements in tactile acuity, proprioceptive measures, and changes in EEG pain biomarkers for the multisensory intervention group only. ConclusionsThe developed multisensory treatment showed a clinically significant reductions in self-reported pain, supported by improvements in objective sensory and neurophysiological measures. These results represent a significant advancement in the treatment and assessment of pain, offering a non-invasive, accessible, and cost-effective solution for neuropathic pain, a major societal burden and one of the most prevalent neurological conditions worldwide. Clinical trial registrationThe trial was registered with ClinicalTrial.gov (NCT05483816).

NFL players have a traumatic injury rate approaching 100%; chronic pain with decreased concentration occur commonly. This study examined the role of a novel focused muscle contraction therapy for the treatment of chronic pain and identified its impact on brain activity. Chronic pain was assessed by numerical score, neuropathic component, and impact on daily activities in 8 retired players. Brain activity was characterized by QEEG with low-resolution electromagnetic tomography analysis and functional measures of visual and auditory attention. Focused muscle contraction muscle therapy administered twice weekly for 6 months was tapered to twice monthly by 12 months. Brodmann Areas (BA) 4 and 9, known to associate with chronic pain, showed values outside the clinically normal range; mean pain duration was 16.5 {+/-} 12.9 years. At 6 months, 5/8 subjects reported pain scores of 0. High beta wave activity was seen in BA 19, 21, 29, 30, and 39, affecting auditory, visual, and body perceptions. Clinically relevant improvements were observed in auditory attention and visual stamina. Pain relief was sustained through 18 months of follow-up. Focused muscle contraction therapy appears to redirect brain activity to new areas of activity which are associated with long-lasting relief of chronic pain and its detriments. This study was registered with clinicaltrial.gov #NCT04822311.

Neuropathic pain (NP) affects approximately 60% of individuals with spinal cord injury (SCI). Existing pharmacological treatments provide only modest relief and are often limited by adverse effects, while non-pharmacological options show small effects at best. As such, there remains a need for accessible, mechanism-informed treatments for SCI-NP. This protocol describes a trial evaluating two promising home-based neuromodulatory interventions for SCI-NP - electroencephalography neurofeedback (EEG-NF) and transcranial direct current stimulation (tDCS) - tested both independently and when applied in combination. We will employ a partially double-blinded (i.e. 1 treatment blinded, the other not), 2x2 factorial randomised controlled trial. Adults with chronic SCI-NP (N=192) will be randomised to: (1) EEG-NF + active tDCS, (2) EEG-NF + sham tDCS, (3) active tDCS alone, or (4) sham tDCS alone, in addition to treatment as usual. Participants will complete 20 home-based sessions over 5 weeks. The primary outcome is change in overall pain severity with the primary endpoint being 6 weeks post-randomisation, with secondary endpoints at 16, 26 and 52 weeks post-randomisation. Secondary outcomes (worst pain intensity, pain interference, sleep, depressive symptoms, health-related quality of life) will be assessed at 6 weeks, 16 weeks, 26 weeks and 52 weeks post-randomisation. This will be the first large-scale trial of home-based EEG-NF and tDCS for SCI-NP. If found to be effective, these scalable interventions could be integrated into routine care and inform further optimisation of neuromodulation strategies for managing SCI-NP.

BackgroundChronic low back pain (CLBP) poses a significant challenge, contributing significantly to the ongoing opioid crisis while also being a leading cause of disability. Although spinal cord stimulation (SCS) stands as the primary FDA-endorsed method for neuromodulatory therapy in CLBP, there remains a subset of patients unresponsive to SCS and others who experience insufficient pain relief over time. In view of the evidence suggesting the critical role of subcallosal cingulate cortex (SCC) connectivity in pain processing, in the current study we investigated the role of the baseline SCC structural as a potential neuroimaging predictive biomarker to identify patients that are likely to benefit from SCS. MethodsDiffusion magnetic resonance imaging scans were acquired in 8 patients with CLBP (mean (SD) age = 70 (10) years; 6 female/2 male, 6 UCLA site, 2 UTSW) before their initial SCS trial. Probabilistic tractography from subject-specific anatomically defined SCC seed regions to the ventral striatum (VS), anterior cingulate cortex (ACC), uncinate fasciculus (UCF) and bilateral medial prefrontal cortex (mPFC) was used to calculate FSL structural probabilistic connectivity in the target network. To explore cross-sectional variations in SCC connectivity related to SCS trial response, we employed a general linear model (GLM) using the SCC probability of connectivity as dependent variable, and the response to the SCS trial as independent variable. We used Pearson correlation to evaluate further the relationships between the critical SCC probability of connectivity and the change in VAS score after the SCS trial. Finally, the role of depression in the treatment outcome was evaluated. ResultsResponders to SCS had significantly lower ipsilateral SCC connectivity to mPFC (F1,8 =8.19, p = 0.03) and VS (F1,8 =17.48, p=0.01) on the left hemisphere compared to non-responders. Pearson correlation analysis showed that decreased ipsilateral SCC baseline connectivity to left mPFC (p=0.03) and VS (p=0.01) was correlated with higher improvement in VAS scores. The baseline depression severity did not significantly influence the change in VAS score following the SCS trial. On the other hand, baseline SCC-VS connectivity on the left hemisphere was a significant predictor of change in VAS score (p=0.02). ConclusionsOur study highlights the important role of SCC connectivity that can serve as a potential biomarker for CLBP stratification and prediction to SCS treatment. These results can reshape our perspective on CLBP management and can serve as early indicator of response to the treatment providing a personalized approach based on the individuals underlying SCC connectivity.

Repetitive transcranial magnetic stimulation (rTMS) of non-motor cortical targets, including the left dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC), and posterosuperior insula (PSI), has been proposed as a treatment for chronic pain with variable clinical outcomes. Pre-treatment local cortical dynamics were hypothesized to serve as markers of chronic pain reduction. In this secondary analysis of a large clinical trial comparing different rTMS targets for pain relief, it was examined whether pre-treatment evoked cortical responses measured by electroencephalography after TMS of DLPFC, ACC, or PSI were associated with at least 30 percent reduction in pain intensity scored on a visual analogue scale. Forty-five patients with chronic pain received 12 sessions over eight weeks of 10 Hz rTMS to DLPFC, ACC or PSI. Cortical reactivity was quantified using global and local mean field power, and oscillatory dynamics were assessed using event-related spectral perturbation (ERSP) and inter-trial coherence (ITC) in the alpha-band (8-12 Hz). Responders (20 of 45, 44%) compared with non-responders showed higher pre-treatment alpha-band ERSP and ITC over the stimulated cortical targets (both P<0.05), and higher alpha-band ERSP and ITC values were negatively correlated with the percentage change in pain intensity (both P < 0.05). These results suggest that elevated pre-treatment TMS-evoked alpha-band oscillatory activity may indicate a higher probability of pain reduction to non-motor rTMS in chronic pain. This supports the development of enrichment strategies using cortical neurophysiology-based markers in neuromodulation trials aimed at individualized, precision-oriented treatments.

NLX-112 is a potent and selective 5-HT1A agonist that has successfully completed phase 2A clinical trial for treatment of L-DOPA-induced dyskinesia in Parkinsons disease (PD). We investigated the neuroprotective activity of NLX-112 in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Four groups of mice received either saline (1ml/kg) daily for 15 days, MPTP (21.4 mg/kg for 5 days, preceded and followed by 5 days saline, NLX-112 (1mg/kg/day for 15 days) or combined MPTP + NLX-112. Two weeks following cessation of treatments, NLX-112-treated mice showed increased locomotor activity and reduced anxiety-like behaviour in an open-field test, consistent with sustained effects of 5-HT1A receptor activation. MPTP-treated male mice showed a significant reduction of dopaminergic (i.e., tyrosine hydroxylase immunoreactive; TH-ir) neurones in the substantia nigra (SN) and the striatum by 40 and 55%, respectively. NLX-112 treatment elicited a significant protection against MPTP-induced loss of TH-ir neurones and nerve terminals. MPTP also a markedly increased the levels of GFAP-ir astrocytes and Iba1-ir microglia in the SN, and co-expression of glial-derived neurotrophic factor (GDNF) in the GFAP-ir astrocytes in both the SN and the striatum. However, in MPTP treated mice, NLX-112, markedly reversed microglial expression in the SN, and upregulated GFAP/GDNF co-localisation in both the striatum and the SN. Overall, the present study demonstrates a robust neuroprotective effect of NLX-112 in a mouse model of PD by preventing microgliosis, upregulating GDNF and favouring sustained pro-locomotor activity.

Parkinsons disease (PD) is the second most common neurodegenerative disease, resulting from accumulation of -synuclein (-syn) in midbrain dopaminergic neurons and progressive neuronal loss. The most relevant species of -syn, oligomers, may exert neurotoxicity in a variety of mechanisms. Accumulation of misfolded -syn in the endoplasmic reticulum (ER) lumen induces ER stress conditions that leads to activation of the Unfolded Protein Response (UPR) and its main sensor PKR-like ER kinase (PERK). PERK is critical for cell fate determination - under prolonged ER stress, it may direct cell towards pro-apoptotic pathways. Targeting of -syn aggregation or UPR by genetic and pharmacological approaches proved effective in preclinical models of PD by previous research. Thus, in the present study, we aimed to determine the potential effect of combination of small-molecule inhibitors of -syn aggregation and ER stress-mediated PERK signaling (namely anle138b and AMG44) in a novel, 3D in vitro model of PD. We demonstrate that combination of both anti-aggregation and ER stress-targeting approaches amplifies neuroprotection against PD in organoid model in terms of increased neuronal metabolic activity, decreased -syn phosphorylation and aggregation, reduced dopaminergic cell death, and restoration of proteostasis.

Parkinsons disease (PD) is becoming increasingly prevalent due to an aging society, which places a substantial disease burden on patients and their families and an annual cost estimated at 52 billion dollars. However, no approved disease modulatory therapies that halt disease progression are available. Alpha-synuclein is a critical therapeutic target found in aggregated form in Lewy bodies which is the diagnostic hallmark of PD. Familial autosomal dominant forms of PD can present with causative exonic point mutations, copy number multiplications, and non-coding risk variants in the alpha-synuclein gene. The disease onset, severity, and progression depend on the gene expression levels of the alpha-synuclein. Here, we demonstrate that Streptococcus aureus dCas9 (sadCas9)-mediated CRISPR interference (CRISPRi) reduces alpha-synuclein mRNA and protein levels in a humanized mouse model. The mechanism of action is based on the principle that a complementary single guide RNA (sgRNA) recruits the sadCas9 protein to the promoter region of alpha-synuclein and modulates target gene transcription, leading to reduced gene expression. We show robust downregulation of alpha-synuclein in neurons after unilateral stereotactic injection into the substantia nigra of adult mice 1 and 6 months after surgery. This work shows proof of concept that viral-mediated sadCas9 CRISPR interference can be a promising therapeutic strategy to reduce alpha-synuclein in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556425v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9247org.highwire.dtl.DTLVardef@beb419org.highwire.dtl.DTLVardef@21e0f9org.highwire.dtl.DTLVardef@391d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisThis study focuses on the development of an AAV9 nuclease-dead S. aureus CRISPR/Cas9 expression system designed to target the human SNCA promoter. The objective is to achieve downregulation of -synuclein (a-syn) expression. After performing surgery and introducing the expression system, the levels of a-syn were measured at 1 month and 6 months post-surgery. The results indicate a significant downregulation of a-syn at both time points. Furthermore, it was observed that the initial immune response to the system attenuated over time, reaching control levels at the 6-month mark. These findings suggest the potential of this expression system for long-term downregulation of a-syn and provide insights into the immune response dynamics associated with its use.

BackgroundChronic pain is a major healthcare problem associated with maladaptive brain circuit changes - many patients are unresponsive to all available therapies. Deep brain stimulation (DBS) is a promising treatment, but traditional targets have not proven consistently effective. DBS for pain may be improved by individualizing location and timing of stimulation based on real time brain measurements and patient reports. MethodsTo optimize personalized stimulation targets, we first performed a double blind, sham-controlled, intracranial EEG brain mapping trial spanning 10 hospital days, in six participants with refractory neuropathic pain syndromes. Five participants with clinically meaningful pain relief were then implanted with permanent devices capable of brain stimulation and recording. We used ambulatory electrical brain recordings to derive bespoke pain biomarkers using machine learning. Pain biomarkers were used in closedloop DBS algorithms for personalized therapy. After an open-label period, we tested the feasibility and efficacy of closed-loop DBS for pain relief against sham in a double-blind, cross-over trial. ResultsTrial intracranial testing revealed multiple brain targets among cortico-striatal-thalamocortical pathways that produced rapid pain relief across participants. We predicted individual pain metrics from both inpatient and ambulatory brain activity with high accuracy; pain biomarkers were incorporated into closed-loop DBS algorithms that also responded to sleep-wake cycles. Personalized, closed-loop DBS was superior to sham, with durability up to 3.5 years. ConclusionsPrecision-medicine DBS, with individually optimized brain stimulation targets and closed-loop delivery of stimulation in response to pain biomarkers, is a feasible strategy to treat refractory chronic pain syndromes. Trial RegistrationNCT04144972

Background: Chronic pain affects millions of patients globally and remains therapeutically chal-lenging. While conventional pharmacological approaches have limitations and side effects, pulsed electromagnetic field (PEMF) therapy represents a non-invasive biophysical approach. However, the biological mechanisms underlying PEMF efficacy remain poorly understood. Objective: This study starting from a multi-center post-market surveillance (PMS) data of 81 patients treated with Synth&eacuteXer (a CE-marked Class IIa PEMF device) proposes a mechanistic framework that links ob- served clinical effects to epigenetic modulation via the histone demethylase KDM6B. Materials and Methods: Patients with inflammatory and degenerative disorders causing chronic pain were treated with Synth&eacuteXer across four Italian rehabilitation centers. Pain was assessed using the Numerical Pain Rating Scale (NPRS) before and after treatment. Statistical analysis included descriptive statistics, ANOVA, correlations, and Cohen d effect size. Proposed mechanisms were based on and extrapolated from molecular and biochemical studies demonstrating KDM6B-dependent epigenetic changes in response to specific PEMF sequences. Results: Mean NPRS score decreased significantly from 8.07 {+/-} 1.65 (PRE) to 1.79 {+/-} 1.67 (POST), representing a 6.28-point reduction (p < 0.001; Cohen d = 3.1). Ninety-eight percent of patients showed pain reduction [&ge;] 2 points. No adverse effects were reported. Subset analysis revealed consistent responses across inflammatory (n=19) and degenerative (n=62) pathologies. Discussion: While the observational nature of these data precludes definitive causal attribution, the magnitude of clinical response combined with emerging evidence of KDM6B-mediated epigenetic remodeling suggests a plausible biological basis for PEMF efficacy. Specifically, sequence-depend- ent electromagnetic stimulation may promote the production of and release of anti-inflammatory cytokines and pain resolution through histone demethylation and chromatin remodeling ultimately acting on the expression modulation of such regulatory cytokines. Conclusions: These post-market surveillance data provide clinical evidence of PEMF effects in chronic pain management. The proposed epigenetic mechanism, while requiring further experimental validation and mechanistic confirmation, offers a science-based framework for understanding PEMF biological action and guiding future investigations.