Microglia, the resident immune cells of the brain, are increasingly recognized as key contributors to Alzheimer’s disease (AD) pathology. Multiple studies have identified microRNA-132 (miR-132) as one of the most significantly downregulated microRNAs in AD. Apart from well-established pleiotropic regulatory functions in neurons, previous evidence also suggested a role for miR-132 in regulating (neuro)inflammation. Yet, the precise mechanisms by which miR-132 impacts microglia remain unknown. In this study, we investigated the role of miR-132 in modulating microglial gene expression and function using gain- and loss-of-function approaches in human-induced pluripotent stem cell (iPSC)-derived microglia (iMGs) from both healthy controls and sporadic AD (sAD) patients. Our findings indicate that while miR-132 may not be indispensable for some baseline microglial functions, increasing its expression in sAD iMGs can reverse disease-associated gene expression changes and attenuate inflammatory responses. To further explore its therapeutic potential, we overexpressed miR-132 in hippocampal neurons of an AD mouse model, employing a clinically relevant adeno-associated viral (AAV) delivery method. miR-132 overexpression was well-tolerated and induced non-cell autonomous effects in microglia. This study sheds light into the regulatory role of miR-132 in microglia under both physiological and AD conditions, and emphasizes the importance of optimizing safe dosage parameters for future clinical applications.

The emergence of functional cerebellar circuits is heavily influenced by activity-dependent processes. However, the contribution of intrinsic Purkinje cell activity to cerebellar development remains less understood. Here, we demonstrate that before synaptic networks mature, Purkinje cell intrinsic activity is essential for regulating dendritic growth, establishing connections with cerebellar nuclei, and ensuring proper cerebellar function. Disrupting this activity during the postnatal period impairs motor function, with earlier perturbations causing more severe deficits. Importantly, only early developmental disruptions lead to pronounced defects in cellular morphology, highlighting key temporal windows for dendritic growth and maturation. Transcriptomic analyses reveal that early intrinsic activity drives the expression of activity-dependent genes, including Prkcg and Car8, which are essential for dendritic development. Our findings emphasize the importance of temporally regulated intrinsic activity in Purkinje cells in guiding cerebellar circuit development, providing a potential unifying mechanism underlying cerebellum-associated disorders.

Mature CNS neurons are incapable of sufficiently regenerating their axons following spinal cord injury (SCI). This is largely due to developmental changes in epigenetic control leading to suppression of axon growth transcriptomic profile, leading to a shift towards synapse function support. Recently, manipulating the PI3K/Akt/mTOR pathway through PI3Kδ overexpression in cortical neurons enhanced axonal regeneration of corticospinal tract axons, which was accompanied by functional recovery monitored for up to 16 weeks. However, PI3K is more widely known for its role as an oncogene, and since overexpression is achieved by the use of AAVs, valid safety concerns are raised as it is unknown what the long-term consequences of sustained PI3Kδ expression in the brain are, which may be necessary to achieve complete re-establishment of the motor pathway. In this study, AAV1-hSYN-PIK3CD was injected into the motor cortex of rats, which survived for 1 year. Comparison with uninjected control animals reveal stable PI3Kδ expression and sustained pathway activation through increased pS6. PI3Kδ-treated animals show absence of tumour formation, neural soma hypertrophy, glial cell activation, or haematological or biochemical abnormalities. Thus, long-term neuronal PI3Kδ expression appears to be well tolerated and may provide a safe and durable strategy to promote functional repair following SCI. ### Competing Interest Statement The authors have declared no competing interest. International Foundation for Research in Paraplegia, https://ror.org/04hhnrj18, P186 Ministry of Education Youth and Sports, https://ror.org/037n8p820, INTER-ACTION-LUAUS25141 Ministry of Education Youth and Sports OPJAK EXREGMED, CZ.02.01.01/00/22_008/0004562

Semaphorin 3A (Sema3A), a known axon chemorepulsive protein during development, is localised in perineuronal nets (PNNs) in the adult brain. PNNs are condensed aggregates of extracellular matrix molecules surrounding specific types of neurons, which regulate neuroplasticity and memory. However, the role of PNN-associated Sema3A in these processes remains unclear. To address this topic, we investigated the contribution of Sema3A to cerebellum-dependent learning and memory in adult mice using the eyeblink conditioning (EBC) paradigm. We interfered with Sema3A signalling by employing: (i) a molecular approach, in which secreted Sema3A receptors (neuropilin-1 bodies) were expressed in the anterior interposed nuclei (AIN) via viral vector injection; and (ii) a genetic approach, using mutant mice with impaired Sema3A signalling (K108N mice). Mice expressing neuropilin-1 bodies showed reduced EBC performance at the beginning of the memory retention phase. However, increased inflammation was found in the AIN of these mice, challenging the interpretation of these findings. K108N mice showed enhanced EBC performance at the beginning of the memory retention phase. No synaptic structural changes were detected in the AIN of K108N mice at the end of the EBC paradigm. Based on our findings in K108N mice, constitutively altered Sema3A signalling is associated with subtle improvement in cerebellar memory.

Introduction:Spinal cord injury involves complex pathobiological mechanisms, necessitating a multidimensional approach for its cure. Previous studies have shown that α9-integrin expression and activation in mature dorsal root ganglion neurons enable the regeneration of injured axons within the spinal cord. However, tissue cavitation and fibrosis impede the regenerating axons from following their usual pathways, forcing them to seek alternative routes rich in tenascin-C, the primary ligand of the integrin. Fibrin gel, an FDA-approved and biocompatible material, can offer three-dimensional support for axonal extension through the cavitated area, thus preventing the formation of aberrant paths and connections that occur in the absence of a suitable scaffold. Methods:The aim of this study was to investigate how combining α9-integrin expression by adeno-associated virus with the use of a fibrin gel as an extracellular microenvironment affects the growth of mature DRG neurites in vitro. Additionally, we sought to functionalize fibrin with integrin ligand peptides, specifically AEIDGIEL, the active domain of tenascin-C, to ensure α9-integrin activation. Results:Our results indicate that fibrin gels are a suitable biomaterial for promoting neurite growth and that AEIDGIEL peptide effectively activates the integrin. Furthermore, we corroborate an autocrine signaling loop of α9-integrin and TN-C produced by neurons. Discussion:the proposed combination therapy of α9-integrin and fibrin gel biomaterials incorporating AEIDGIEL peptide shows promise for addressing the complex challenges of spinal cord injury and promoting effective neural regeneration, laying the foundation for further in vivo research.

Multiple sclerosis (MS) is a neurological disease characterized by demyelinating lesions in the CNS. This study investigated whether a minimally invasive adeno-associated virus (AAV) vector (AAV.PHP.eB) can direct transgene expression in CNS cell types relevant to MS, including astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), microglia, and neurons in experimental autoimmune encephalitis, a widely used MS model. In vivo bioluminescence imaging and histological analysis following AAV.PHP.eB-mediated gene delivery in healthy mice using the ubiquitous CAG promoter and five neural promoters (MBP, Sox10, hSyn1, gfa2, and gfaABC1D) revealed long-term, robust, and cell-type-specific activity across the brain and spinal cord. AAV.PHP.eB is capable of traversing the blood-brain barrier in experimental autoimmune encephalitis (EAE) and directs sustained and cell-type-specific transgene expression for the MBP, Sox10, hSyn1, and gfaABC1D promoters. The MBP and Sox10 promoters directed transgene expression in oligodendroglia around and within inflammatory demyelinating lesions, whereas the gfaABC1D promoter directs transgene expression in gray and white matter astrocytes and hSyn1 in neurons. The neural promoters were minimally active in the periphery, with the exception of gfa2. This methodological study is a first step toward the development of minimally invasive gene therapy to promote myelin repair and/or suppress inflammation in MS.

Factor VII (FVII) deficiency is a rare bleeding disorder with a prevalence of approximately 1:300,000-500,000 individuals. We explored whether adeno-associated virus (AAV)-mediated gene therapy can achieve durable and functional expression of human FVII (hFVII) in vivo. Wild-type hFVII (hFVIIwt) and a naturally occurring splice variant designated hFVII(-22) (GenBank: NM_019616.4) were expressed under the control of the Apolipoprotein E-derived hepatic locus control region and the α1-anti-trypsin promoter. Expression cassettes were packaged in either recombinant AAV5 (rAAV5) or recombinant AAV8 (rAAV8). For both hFVIIwt and hFVII(-22), 5- to 20-fold higher plasma levels of hFVII could be obtained when rAAV8 was used as a vector as opposed to rAAV5. Interestingly, hFVII levels obtained by employing rAAV8 expressing the hFVII(-22) cDNA variant were approximately 10 times higher than those obtained using rAAV8 expressing hFVIIwt. Based on these results, we generated an rAAV8-based gene therapy vector encoding hFVII(-22) and evaluated long-term expression in vivo. Employing a vector dose of 0.8 × 1012 genome copies (gc)/kg, we observed 48 weeks of functional hFVII expression which peaked at 16 IU/mL and stabilized at 7 IU/mL. These results support the pre-clinical development of AAV8-mediated delivery employing the splice-variant hFVII(-22) for patients suffering from FVII deficiency.

Functional recovery after spinal cord injury (SCI) is hindered by the limited ability of axons to regenerate in the adult mammalian central nervous system (CNS). Overcoming this barrier is critical for achieving effective recovery. Axonal regeneration depends on the activation of intracellular processes like transcription factor induction, protein and lipid trafficking, and cytoskeletal remodelling. Targeting these pathways offers a promising approach for promoting neuronal repair. This study examined the combined therapeutic effects of dibutyryl-cAMP (db-cAMP), which primes neurons for growth, and integrin α9 overexpression, which supports axonal extension. Using in vitro models with dorsal root ganglion (DRG) neurons and astrocytes, as well as an in vivo SCI model, we evaluated the potential of this approach. In vitro, the combination of db-cAMP and integrin α9 significantly enhanced neuronal growth. However, in vivo results were less consistent, with db-cAMP affecting AAV-mediated transcription and the expression of tenascin C (TnC) in neurons and astrocytes. These findings highlight the potential of modulating intracellular signalling and integrin activation but underscore the challenges posed by the complexity of the in vivo environment. Further studies are necessary to unravel these mechanisms and refine therapeutic strategies for effective SCI recovery. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Czech Science Foundation, 24-11193S Ministry of Education, Youth and Sports, Excellence in Regenerative Medicine; CZ.02.01.01/00/22_008/0004562 Charles University Grant Agency, 320421, 102122 [1]: pending:yes

Multiple sclerosis (MS) is a highly heterogeneous disease, with varying remyelination potential across individuals and between lesions. However, the molecular mechanisms underlying the potential to remyelinate remain poorly understood. In this study, we aimed to take advantage of the intrinsic heterogeneity in remyelinating capacity between MS donors and lesions to uncover known and novel pro-remyelinating molecules for MS therapies. To elucidate distinct molecular signatures underlying the potential to remyelinate, we stratified MS donors from the Netherlands Brain Bank cohort (n = 239), based on proportions of remyelinated lesions (RLs), into efficiently remyelinating donors (ERDs; n = 21) and poorly remyelinating donors (PRDs; n = 19). We performed bulk RNA sequencing of RLs, active lesions with ramified and amoeboid microglia/macrophage morphology (ALs non-foamy), active lesions with foamy microglia/macrophage morphology (ALs foamy) and normal-appearing white matter (NAWM) from ERDs and PRDs. We found that ALs non-foamy were positively correlated with remyelination, whereas ALs foamy were not, indicating a role for microglia/macrophage state in influencing remyelination potential. Bioinformatics analyses were performed to identify key pathways and molecules implicated in the remyelination process. We found distinct differences between the donors with differing remyelination potential in comparable MS lesion types. The RLs and ALs non-foamy of ERDs versus PRDs showed upregulation of the epithelial-mesenchymal transition pathway, whereas in ALs foamy of PRDs, inflammation and damage-associated pathways (i.e. MTORC1 signalling, TNF signalling and oxidative phosphorylation) were upregulated in comparison to ALs foamy of ERDs, suggesting that these latter pathways might counteract remyelination. We found genes significantly upregulated in RLs and/or ALs non-foamy of ERDs that have previously been associated with remyelination, including CXCL12, EGF, HGF, IGF2, IL10, PDGFB, PPARG and TREM2, illustrating the strength of our donor and lesion stratification. TGFB1, TGFB2, EGF and IGF1 were determined to be key upstream regulators of genes upregulated in RLs and ALs non-foamy of ERDs. We also identified potential novel pro-remyelinating molecules, such as BTC, GDF10, GDF15, CCN1, CCN4, FGF5, FGF10 and INHBB. Our study identified both known and novel genes associated with efficient remyelination that might facilitate the development of therapeutic strategies to promote tissue repair and clinical recovery in MS.

Full recovery from spinal cord injury requires axon regeneration to re-establish motor and sensory pathways. In mammals, the failure of sensory and motor axon regeneration has many causes intrinsic and extrinsic to neurons, amongst which is the lack of adhesion molecules needed to interact with the damaged spinal cord. This study addressed this limitation by expressing the integrin adhesion molecule α9, along with its activator kindlin-1, in sensory neurons via adeno-associated viral (AAV) vectors. This enabled sensory axons to regenerate through spinal cord injuries and extend to the brainstem, restoring sensory pathways, touch sensation and sensory behaviours. One of the integrin ligands in the injured spinal cord is tenascin-C, which serves as a substrate for α9β1 integrin, a key receptor in developmental axon guidance. However, the adult PNS and CNS neurons lack this receptor. Sensory neurons were transduced with α9 integrin (which pairs with endogenous β1 to form a α9β1 tenascin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with α9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4,5 sensory ganglia injected with AAV1 vectors. In animals treated with α9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesions and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Axons grew through the dorsal grey matter rather than their normal pathway the dorsal columns. Growth was slow, axons taking 12 weeks to grow from T10 to the medulla, a distance of 4–5 cm. Functional recovery was confirmed through cFos activation in neurons rostral to the injury after nerve stimulation and VGLUT1/2 staining indicating new synapse formation above the lesion. Behavioural recovery was seen in both heat and mechanical sensation, as well as tape removal tests. This approach demonstrates the potential of integrin-based therapies for long distance sensory axon regeneration and functional recovery following thoracic and partial recovery after cervical spinal cord injury.

Adaptive behavioral responses to stressors are critical for survival. However, which brain areas orchestrate switching the appropriate stress responses to distinct contexts is an open question. This study aimed to identify the cell-type-specific brain circuitry governing the selection of distinct behavioral strategies in response to stressors. Through novel mouse behavior paradigms, we observed distinct stressor-evoked behaviors in two psycho-spatially distinct contexts characterized by stressors inside or outside the safe zone. The identification of brain regions activated in both conditions revealed the involvement of the dorsomedial hypothalamus (DMH). Further investigation using optogenetics, chemogenetics, and photometry revealed that glutamatergic projections from the DMH to periaqueductal gray (PAG) mediated responses to inside stressors, while GABAergic projections, particularly from tachykinin1-expressing neurons, played a crucial role in coping with outside stressors. These findings elucidate the role of cell-type-specific circuitry from the DMH to the PAG in shaping behavioral strategies in response to stressors. These findings have the potential to advance our understanding of fundamental neurobiological processes and inform the development of novel approaches for managing context-dependent and anxiety-associated pathological conditions such as agoraphobia and claustrophobia.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100 -fold higher titer (minimally 1 x 10 11 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material -intensive process. This constraint prompted the development of a protocol for suspension -based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 10 12 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 10 12 GC in total, and TransIt yielded 2.4 x 10 13 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Selecting the right response to emotional stressors is vital for survival. From the psycho-spatial point of view, the perceived stressors can fall into two conditions: the stressors outside the confined safe zone or inside. Although this novel approach of categorizing the stressors distinguishes two anxiety-associated pathological conditions, agoraphobia and claustrophobia, the difference between the behavioral responses in these two conditions and the underlying brain circuits are not well understood. Here, using novel behavioral paradigms in mice, we found that outside- and inside-stressors elicit two distinct behaviors, push-bedding and escape-jumping, respectively. Next, in an attempt to identify the brain regions activated in both outside- and inside-stressor conditions, the whole-brain c-Fos staining led us to an unexpected brain region, dorsomedial hypothalamus (DMH). Using optogenetics, chemogenetics, and photometry recording, we discovered that glutamatergic projections from DMH to the periaqueductal gray drive the escape to the inside-stressor, while GABAergic projections, in particular from neurons expressing tachykinin 1, are essential for coping with the outside-stressor. We conclude that distinct populations within the DMH region control the response to psycho-spatially distinct stressors. This discovery shows how the stressors and the spatial information of the stressors integrate in the brain and it is the first to shed light on understanding the narrow border between the two space-associated stress disorders/phobias, i.e. agoraphobia and claustrophobia and the underlying brain mechanisms.

Full recovery from spinal cord injury can only occur if the axon pathways connecting the brain and spinal cord regenerate and restore motor and sensory connections. Neither sensory nor motor axons can regenerate spontaneously in the spinal cord in mammals. This failure is partly due to the lack of suitable adhesion molecules on the sensory axons that allows them to interact with the environment of the damaged spinal cord. In this rat study, an integrin adhesion molecule along with its activator was expressed in sensory neurons using an adeno-associated viral (AAV) vector. Expression of these adhesion molecules allowed sensory axons to regenerate through the spinal cord injury and all the way back to the brainstem, restoring the sensory pathway. Treated animals regained touch sensation and sensory behaviours. The integrin ligands in the injured spinal cord are tenascin-C and osteopontin, but adult PNS and CNS neurons lack receptors to them. Sensory neurons were transduced with α9 integrin, which combines with endogenous β1 as α9β1 (which is a tenascin/osteopontin receptor) together with the integrin activator kindlin-1. Regeneration from sensory neurons transduced with α9integrin and kindlin-1 was examined after C4 and after T10 dorsal column lesions with C6,7 and L4.5 sensory ganglia injected with AAV1 vectors. In animals treated with α9 integrin and kindlin-1, sensory axons regenerated through tenascin-C-expressing connective tissue strands and bridges across the lesion and then re-entered the CNS tissue. Many axons regenerated rostrally to the level of the medulla. Regenerated axons were particularly visible at the border between white and grey matter in the dorsal cord. Stimulation of the median/sciatic nerve caused many neurons rostral to the injury to activate and express cFos. VGLUT1/2 staining indicated newly formed functional synapses above the lesion. Behavioural recovery was seen in heat, mechanical sensation and tape removal tests. Many axons regenerated from the thoracic lesions to the brainstem, a distance of 4-5 cm, equivalent to the length of 1 or 2 spinal segments in humans.

Cervical level spinal cord injury (SCI) can severely impact upper limb muscle function, which is typically assessed in the clinic using electromyography (EMG). Here, we established novel preclinical methodology for EMG assessments of muscle function after SCI in awake freely moving animals. Adult female rats were implanted with EMG recording electrodes in bicep muscles and received bilateral cervical (C7) contusion injuries. Forelimb muscle activity was assessed by recording maximum voluntary contractions during a grip strength task and cortical motor evoked potentials in the biceps. We demonstrate that longitudinal recordings of muscle activity in the same animal are feasible over a chronic post-injury time course and provide a sensitive method for revealing post-injury changes in muscle activity. This methodology was utilized to investigate recovery of muscle function after a novel combination therapy. Cervical contused animals received intraspinal injections of a neuroplasticity-promoting agent (lentiviral-chondroitinase ABC) plus 11 weeks of cortical epidural electrical stimulation (3 h daily, 5 days/week) and behavioral rehabilitation (15 min daily, 5 days/week). Longitudinal monitoring of voluntary and evoked muscle activity revealed significantly increased muscle activity and upper limb dexterity with the combination treatment, compared to a single treatment or no treatment. Retrograde mapping of motor neurons innervating the biceps showed a predominant distribution across spinal segments C5-C8, indicating that treatment effects were likely due to neuroplastic changes in a mixture of intact and injured motor neurons. Thus, longitudinal assessments of muscle function after SCI correlate with skilled reach and grasp performance and reveal functional benefits of a novel combination therapy.

Aging is associated with increased prevalence of axonal injuries characterized by poor regeneration and disability. However, the underlying mechanisms remain unclear. In our experiments, RNA sequencing of sciatic dorsal root ganglia (DRG) revealed significant aging-dependent enrichment in T cell signaling both before and after sciatic nerve injury (SNI) in mice. Lymphotoxin activated the transcription factor NF-κB, which induced expression of the chemokine CXCL13 by neurons. This in turn recruited CXCR5+CD8+ T cells to injured DRG neurons overexpressing major histocompatibility complex class I. CD8+ T cells repressed the axonal regeneration of DRG neurons via caspase 3 activation. CXCL13 neutralization prevented CXCR5+CD8+ T cell recruitment to the DRG and reversed aging-dependent regenerative decline, thereby promoting neurological recovery after SNI. Thus, axonal regeneration can be facilitated by antagonizing cross-talk between immune cells and neurons.