Search

We describe here an innovative, non-transgenic animal model of Alzheimer's disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimer's disease. After 2 weeks of treatment, we detected progressive Alzheimer's disease-like neuropathology, with an increase of β-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment. [ABSTRACT FROM AUTHOR]

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).Cell Death and Differentiation (2007) 14, 240–253. doi:10.1038/sj.cdd.4401988; published online 23 June 2006 [ABSTRACT FROM AUTHOR]

The c-Jun N-terminal kinases (JNKs) are a family of proteins that, once activated by stress stimuli, can alter neuronal functions and survival. The JNK cascade plays a crucial role in the post-synaptic neuronal compartment by altering its structural organization and leading, at worst, to an overall impairment of neuronal communication. Increasing evidence suggests that synaptic impairment is the first neurodegenerative event in Alzheimer's disease (AD). To better elucidate this mechanism, we longitudinally studied 5xFAD mice at three selected time points representative of human AD symptom progression. We tested the mice cognitive performance by using the radial arm water maze (RAWM) in parallel with biochemical evaluations of post-synaptic enriched protein fraction and total cortical parenchyma. We found that 5xFAD mice presented a strong JNK activation at 3.5 months of age in the post-synaptic enriched protein fraction. This JNK activation correlates with a structural alteration of the post-synaptic density area and with memory impairment at this early stage of the disease that progressively declines to cause cell death. These findings pave the way for future studies on JNK as a key player in early neurodegeneration and as an important therapeutic target for the development of new compounds able to tackle synaptic impairment in the early phase of AD pathology.

Competing Interests: None

Introduction: Autism spectrum disorders (ASD) are the most prevalent neurobiological disorders in children. The etiology comprises genetic, epigenetic, and environmental factors such as dysfunction of the immune system. Epigenetic mechanisms are mainly represented by DNA methylation, histone modifications, and microRNAs (miRNA). The major explored epigenetic mechanism is mediated by miRNAs which target genes known to be involved in ASD pathogenesis. Salivary poly-omic RNA measurements have been associated with ASD and are helpful to differentiate ASD endophenotypes. This study aims to comprehensively examine miRNA expression in children with ASD and to reveal potential biomarkers and possible disease mechanisms so that they can be used to improve faction between individuals by promoting more personalized therapeutic approaches., Materials and Methods: Saliva samples were collected from 10 subjects: 5 samples of children with ASD and 5 from healthy controls. miRNAs were analyzed using an Illumina Next-Generation-Sequencing (NGS) system., Results: Preliminary data highlighted the presence of 365 differentially expressed miRNAs. Pathway analysis, molecular function, biological processes, and target genes of 41 dysregulated miRNAs were assessed, of which 20 were upregulated, and 21 were downregulated in children with ASD compared to healthy controls., Conclusion: The results of this study represent preliminary but promising data, as the identified miRNA pathways could represent useful biomarkers for the early non-invasive diagnosis of ASD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kalemaj, Marino, Santini, Tomaselli, Auti, Cagetti, Borsello, Costantino, Inchingolo, Boccellino, Di Domenico and Tartaglia.)

The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases.

c-Jun N-terminal kinases (JNKs) are stress-activated serine/threonine protein kinases belonging to the mitogen-activated protein kinase (MAPK) family. Among them, JNK3 is selectively expressed in the central nervous system, cardiac smooth muscle, and testis. In addition, it is the most responsive JNK isoform to stress stimuli in the brain, and it is involved in synaptic dysfunction, an essential step in neurodegenerative processes. JNK3 pathway is organized in a cascade of amplification in which signal transduction occurs by stepwise, highly controlled phosphorylation. Since different MAPKs share common upstream activators, pathway specificity is guaranteed by scaffold proteins such as JIP1 and β-arrestin2. To better elucidate the physiological mechanisms regulating JNK3 in neurons, and how these interactions may be involved in synaptic (dys)function, we used (i) super-resolution microscopy to demonstrate the colocalization among JNK3-PSD95-JIP1 and JNK3-PSD95-β-arrestin2 in cultured hippocampal neurons, and (ii) co-immunoprecipitation techniques to show that the two scaffold proteins and JNK3 can be found interacting together with PSD95. The protein-protein interactions that govern the formation of these two complexes, JNK3-PSD95-JIP1 and JNK3-PSD95-β-arrestin2, may be used as targets to interfere with their downstream synaptic events.

Type 2 diabetes (T2D) is associated with multiple comorbidities, including diabetic retinopathy (DR) and cognitive decline, and T2D patients have a significantly higher risk of developing Alzheimer's disease (AD). Both DR and AD are characterized by a number of pathological mechanisms that coalesce around the neurovascular unit, including neuroinflammation and degeneration, vascular degeneration, and glial activation. Chronic hyperglycemia and insulin resistance also play a significant role, leading to activation of pathological mechanisms such as increased oxidative stress and the accumulation of advanced glycation end-products (AGEs). Understanding these common pathways and the degree to which they occur simultaneously in the brain and retina during diabetes will provide avenues to identify T2D patients at risk of cognitive decline., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Rett syndrome (RTT) is a monogenic X-linked neurodevelopmental disorder characterized by loss-of-function mutations in the MECP2 gene, which lead to structural and functional changes in synapse communication, and impairments of neural activity at the basis of cognitive deficits that progress from an early age. While the restoration of MECP2 in animal models has been shown to rescue some RTT symptoms, gene therapy intervention presents potential side effects, and with gene- and RNA-editing approaches still far from clinical application, strategies focusing on signaling pathways downstream of MeCP2 may provide alternatives for the development of more effective therapies in vivo. Here, we investigate the role of the c-Jun N-terminal kinase (JNK) stress pathway in the pathogenesis of RTT using different animal and cell models and evaluate JNK inhibition as a potential therapeutic approach., Results: We discovered that the c-Jun N-terminal kinase (JNK) stress pathway is activated in Mecp2-knockout, Mecp2-heterozygous mice, and in human MECP2-mutated iPSC neurons. The specific JNK inhibitor, D-JNKI1, promotes recovery of body weight and locomotor impairments in two mouse models of RTT and rescues their dendritic spine alterations. Mecp2-knockout presents intermittent crises of apnea/hypopnea, one of the most invalidating RTT pathological symptoms, and D-JNKI1 powerfully reduces this breathing dysfunction. Importantly, we discovered that also neurons derived from hiPSC-MECP2 mut show JNK activation, high-phosphorylated c-Jun levels, and cell death, which is not observed in the isogenic control wt allele hiPSCs. Treatment with D-JNKI1 inhibits neuronal death induced by MECP2 mutation in hiPSCs mut neurons., Conclusions: As a summary, we found altered JNK signaling in models of RTT and suggest that D-JNKI1 treatment prevents clinical symptoms, with coherent results at the cellular, molecular, and functional levels. This is the first proof of concept that JNK plays a key role in RTT and its specific inhibition offers a new and potential therapeutic tool to tackle RTT., (© 2021. The Author(s).)

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

During Central Nervous System ontogenesis, myelinating oligodendrocytes (OLs) arise from highly ramified and proliferative precursors called oligodendrocyte progenitor cells (OPCs). OPC architecture, proliferation and oligodendro-/myelino-genesis are finely regulated by the interplay of cell-intrinsic and extrinsic factors. A variety of extrinsic cues converge on the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway. Here we found that the germinal ablation of the MAPK c-Jun N-Terminal Kinase isoform 1 (JNK1) results in a significant reduction of myelin in the cerebral cortex and corpus callosum at both postnatal and adult stages. Myelin alterations are accompanied by higher OPC density and proliferation during the first weeks of life, consistent with a transient alteration of mechanisms regulating OPC self-renewal and differentiation. JNK1 KO OPCs also show smaller occupancy territories and a less complex branching architecture in vivo. Notably, these latter phenotypes are recapitulated in pure cultures of JNK1 KO OPCs and of WT OPCs treated with the JNK inhibitor D-JNKI-1. Moreover, JNK1 KO and WT D-JNKI-1 treated OLs, while not showing overt alterations of differentiation in vitro, display a reduced surface compared to controls. Our results unveil a novel player in the complex regulation of OPC biology, on the one hand showing that JNK1 ablation cell-autonomously determines alterations of OPC proliferation and branching architecture and, on the other hand, suggesting that JNK1 signaling in OLs participates in myelination in vivo.

SUMOylation of proteins plays a key role in modulating neuronal function. For this reason, the balance between protein SUMOylation and deSUMOylation requires fine regulation to guarantee the homeostasis of neural tissue. While extensive research has been carried out on the localization and function of small ubiquitin-related modifier (SUMO) variants in neurons, less attention has been paid to the SUMO-specific isopeptidases that constitute the human SUMO-specific isopeptidase (SENP)/Ubiquitin-Specific Protease (ULP) cysteine protease family (SENP1-3 and SENP5-7). Here, for the first time, we studied the localization of SENP1, SENP6, and SENP7 in cultured hippocampal primary neurons at a super resolution detail level, with structured illumination microscopy (SIM). We found that the deSUMOylases partially colocalize with pre- and post-synaptic markers such as synaptophysin and drebrin. Thus, further confirming the presence with synaptic markers of the negative regulators of the SUMOylation machinery.

The c-Jun N -terminal kinase 3 (JNK3) is the JNK isoform mainly expressed in the brain. It is the most responsive to many stress stimuli in the central nervous system from ischemia to Aβ oligomers toxicity. JNK3 activity is spatial and temporal organized by its scaffold protein, in particular JIP-1 and β-arrestin-2, which play a crucial role in regulating different cellular functions in different cellular districts. Extensive evidence has highlighted the possibility of exploiting these adaptors to interfere with JNK3 signaling in order to block its action. JNK plays a key role in the first neurodegenerative event, the perturbation of physiological synapse structure and function, known as synaptic dysfunction. Importantly, this is a common mechanism in many different brain pathologies. Synaptic dysfunction and spine loss have been reported to be pharmacologically reversible, opening new therapeutic directions in brain diseases. Being JNK3-detectable at the peripheral level, it could be used as a disease biomarker with the ultimate aim of allowing an early diagnosis of neurodegenerative and neurodevelopment diseases in a still prodromal phase.

Deficiency of the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while higher levels are linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A loss or gain of function in these disorders are still not well understood, and treatments are still lacking. Here, using the Ube3a maternal loss (Ube3a m-/p+ ) mouse model, we report an important JNK signaling activation in the hippocampus, cortex and cerebellum correlating with the onset of behavioral defects and biochemical marker alterations in the post-synaptic element, suggesting important spine pathology. JNK activation occurs at 7 and persists up till 23 weeks in Ube3a m-/p+ mice in two different cellular compartments: the nucleus and the post-synaptic protein-enriched fraction. To study JNK's role in Ube3a m-/p+ pathology we treated mice with the specific JNK inhibitor peptide, D-JNKI1, from 7 to 23 weeks of age. Preventing JNK action in vivo restores the post-synaptic protein-enriched fraction defects and the cognitive impairment in these mice. Our results imply a critical role of UBE3A-JNK signaling in the pathogenesis of UBE3A-related disorders. In particular, it was clear that JNK is a key player in regulating AS synaptic alterations and the correlated cognitive impairments, in fact, its specific inhibition tackles Ube3a m-/p+ pathology. This study sheds new light on the neuronal functions of UBE3A and offers new prospects for understanding the pathogenesis of UBE3A-related disorders., Competing Interests: Declaration of Competing Interest The authors declare no actual or potential conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

While protein synthesis in neurons is largely attributed to cell body and dendrites, the capability of synaptic regions to synthesize new proteins independently of the cell body has been widely demonstrated as an advantageous mechanism subserving synaptic plasticity. Thus, the contribution that local protein synthesis at synapses makes to physiology and pathology of brain plasticity may be more prevalent than initially thought. In this study, we tested if local protein synthesis at synapses is deregulated in the brains of TgCRND8 mice, an animal model for Alzheimer's disease (AD) overexpressing mutant human amyloid precursor protein (APP). To this end, we used synaptosomes as a model system to study the functionality of the synaptic regions in mouse brains. Our results showed that, while TgCRND8 mice exhibit early signs of brain inflammation and deficits in learning, the electrophoretic profile of newly synthesized proteins in their synaptosomes was subtly different from that of the control mice. Interestingly, APP itself was, in part, locally synthesized in the synaptosomes, underscoring the potential importance of local translation at synapses. More importantly, after the contextual fear conditioning, de novo synthesis of some individual proteins was significantly enhanced in the synaptosomes of control animals, but the TgCRND8 mice failed to display such synaptic modulation by training. Taken together, our results demonstrate that synaptic synthesis of proteins is impaired in the brain of a mouse model for AD, and raise the possibility that this deregulation may contribute to the early progression of the pathology.

Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson's disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an "upstream" oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis.

Pathological Tau (P-Tau) leads to dementia and neurodegeneration in tauopathies, including Alzheimer's disease. The P301L transgenic mice well mimic human tauopathy features; P-Tau localizes also at the dendritic spine level and this correlates with synaptic markers down-regulation. Importantly, tg females present a more severe pathology compared to male mice. We describe JNK activation in P301L-tg mice, characterizing by P-JNK and P-c-Jun, cleaved-Caspase-3, P-PSD95 and P-Tau (direct JNK-targets) increased levels in tg vs control mice. These data indicate that JNK stress pathway is involved in neuronal degenerative mechanisms of this mouse model. In addition, P-JNK level is higher in females compared to male tg mice, underlying a sexual dimorphism in the JNK pathway activation. The behavioral studies highlight that tg females present major cognitive and locomotor defects, strongly correlated with a more severe synaptic injury, in comparison to tg male. Notably, at the dendritic spine level, JNK is powerfully activated and its level reveals a sexual dimorphism that is coherent with behavioral defects and spine pathology. The P301L's synaptic pathology is characterized by a strong increase of P-PSD95/PSD95 and P-JNK/JNK ratios and by an augmented level of cleaved-Caspase-3 and a decrease of Drebrin level in the post-synaptic elements. These results suggest that JNK plays a key role in synaptopathy of P301L mice. Importantly, until now, there are any efficient treatments against synaptic pathology and JNK could represent an interesting target to tackle P-Tau-induced synaptic pathology. It will be important to test specific JNK inhibitors to verify their potential neuroprotective effect., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Spinal muscular atrophy (SMA) is a severe neurodegenerative disorder that occurs in early childhood. The disease is caused by the deletion/mutation of the survival motor neuron 1 (SMN1) gene resulting in progressive skeletal muscle atrophy and paralysis, due to the degeneration of spinal motor neurons (MNs). Currently, the cellular and molecular mechanisms underlying MN death are only partly known, although recently it has been shown that the c-Jun NH 2 -terminal kinase (JNK)-signaling pathway might be involved in the SMA pathogenesis. After confirming the activation of JNK in our SMA mouse model (SMN2+/+; SMNΔ7+/+; Smn-/-), we tested a specific JNK-inhibitor peptide (D-JNKI1) on these mice, by chronic administration from postnatal day 1 to 10, and histologically analyzed the spinal cord and quadriceps muscle at age P12. We observed that D-JNKI1 administration delayed MN death and decreased inflammation in spinal cord. Moreover, the inhibition of JNK pathway improved the trophism of SMA muscular fibers and the size of the neuromuscular junctions (NMJs), leading to an ameliorated innervation of the muscles that resulted in improved motor performances and hind-limb muscular tone. Finally, D-JNKI1 treatment slightly, but significantly increased lifespan in SMA mice. Thus, our results identify JNK as a promising target to reduce MN cell death and progressive skeletal muscle atrophy, providing insight into the role of JNK-pathway for developing alternative pharmacological strategies for the treatment of SMA.