Synaptic alterations as a common phase in neurology and... : Research in neural regeneration (2023)

Brain synapses play a key role in neuronal communication: this 'conversation' is the basis of all brain activity, and synaptic dysfunction leads to brain disorders. We study the modulators of this critical synaptic function and present here the evidence supporting the c-Jun N-terminal kinase (JNK) pathway as a central player in this scenario.

The term "synapse" comes from the Greekmore('together') and ('touch', 'connection') meaning 'connection'. Synapses are highly specialized cellular connections that represent the basic building blocks of neuronal communication.

There are two different types of synapses: In electrical synapses, the presynaptic and postsynaptic cell membranes are connected by special junctions called gap junctions, channels that allow ions to pass from one neuron to another. At chemical synapses, which represent the majority of synapses in the mammalian nervous system, electrical activity in the presynaptic neuron is converted into the release of a chemical neurotransmitter that binds to receptors located on the plasma membrane of the postsynaptic cell. Therefore, two separate cellular components make up the chemical synapse: the presynaptic component, which specializes in the release of neurotransmitters, and the postsynaptic component, which binds to chemical compounds, converting them into an electrical signal. Presynaptic and postsynaptic elements are linked and reflect changes to each other, so that changes in one element correspond to a change in the second.

What was described above defines the ancient "bilateral" synapse, more recently updated by the addition of a third building block: tiny astrocytic processes. This astrocytic component responds to synaptic activity and, in turn, participates in the regulation of synaptic transmission. Now we also look at a fourth type of cell, microglia, which makes brief, repetitive contacts with synapses. These dynamic interactions occur in health, but also in pathological conditions such as Alzheimer's disease (AD), where microglia and immune molecules participate in eliminating dysfunctional synapses (Wilton et al., 2019).

Synapses, both excitatory and inhibitory, are critical in shaping brain function and undergo dynamic changes. Dynamic changes of synapses are mainly studied by visualizing the postsynaptic structure called spine. In fact, by monitoring the spines in the living brain it was discovered that they were maintained in a state of equilibrium that guaranteed their continuous formation and elimination, resulting in a decrease or increase in column rotation or a temporary shift to facilitate them. additions or deletions (Holtmaat et al., 2005).

Therefore, it is very important to keep in mind that a single spine is constantly evolving. it changes shape according to the needs and activities of presynaptic and postsynaptic neurons and the neural network. This phenomenon is known as synaptic plasticity, a very important characteristic of inhibitory and excitatory neurons, through which they can adapt to environmental changes. Inhibitory synapses are difficult to image and less studied, resulting in poorly understood biological changes compared to excitatory synapses. However, inhibitory synapses are four times more dynamic than their axon counterparts (Chen et al., 2012).

Brain plasticity is essential to maintain its adaptive functionality underlying learning and memory, but stressful stimuli can also induce structural changes at the level of synapses. Increasing evidence indicates that, in brain diseases, the first neurodegenerative mechanism occurs at the synapse. Therefore, it is important to understand the intracellular mechanisms underlying synaptic modulation to design potent neuroprotective strategies.

With this in mind, we decided to examine synaptic dysfunction as a common alteration of neurobiological mechanisms, from neurodevelopmental (Angelman and Rett syndromes) to chronic neurodegenerative diseases (Alzheimer and Taoopathy).

Plasticity is a central feature of the developing and adult brain. Much evidence shows that neurodevelopmental disorders, particularly autism spectrum disorders (among them Angelman, Rett and Dravet syndromes, Fragile X, etc.), are characterized by too many synaptic connections causing hyperconnectivity of brain circuits. In contrast, studies of aging and neurodegenerative brain diseases revealed an opposite trend with loss of synaptic connectivity (Penzes et al., 2011).

In this context, synaptic dysfunction, which usually precedes neuronal death, has been associated with many neurodegenerative diseases, such as Parkinson's disease, Huntington's disease, and AD, as well as neuropsychiatric diseases. Many mutations in the human synapse proteome (the “synapse”) have been described as underlying psychiatric and neurological disorders.

Synaptic dysfunction is an emerging hypothesis to explain emotional disorders, and it has been shown that loss of synaptic homeostasis in specific networks also contributes to chronic pain (Torres et al., 2017). Furthermore, a network-level imbalance between excitatory and inhibitory synaptic signals causes epilepsy, and such an imbalance has also been shown in neurodevelopmental disorders such as Rett and Angelman syndromes and other autism spectrum disorders (Canitano and Palumbi, 2021).

Although brain disorders have many different pathologic/clinical manifestations as well as causes (ie, cerebral ischemia: interruption of blood flow, Parkinson's disease: degeneration of dopaminergic neurons in the substantia nigra) mediated by different intracellular molecular pathways, they all share synaptopathy.

Therefore, a better understanding of the mechanisms of synaptic dysfunction will pave the way for new exploitable therapeutic strategies in many different brain diseases, thus promoting neuroprotection against aberrant neurodegenerative/neurodevelopmental mechanisms.

Thus, the central focus of our research is the study of key proteins in synaptic dysfunction/dysmorphogenesis. These early synaptic changes represent a promising therapeutic strategy and a critical time window for neuroprotective therapy. In fact, synapses dynamically change and respond to stress stimuli, passing through an initial reversible phase, in which synaptic function is impaired, but can be rescued to a second phase, if the stress persists, where synaptic damage is maintained. becomes irreversible and progresses to synaptic death. loss and ultimately neuronal death.

Here we report our studies on neurodevelopment (Angelman and Rett syndromes) as well as neurodegenerative diseases (Alzheimer and Tauopathy) (Sclip et al., 2014; Buccarello et al., 2018; Musi et al., 2020, 2021). Previous evidence from our work has shown that the JNK signaling pathway is involved in the stress response, as well as in other diverse physiological functions, and is a common factor in the synaptopathy of all these brain diseases.

Why is JNK a key player in neurodegenerative mechanisms? Because JNK regulates 3 main actions in neurons which are highly polarized cells. Indeed, JNK performs several functions in different cellular compartments: 1 - in the presynaptic terminal, it regulates vesicle closure and neurotransmitter release (Biggi et al., 2017); 2 - at the postsynaptic level, it controls the correct organization of receptors and scaffolding proteins in the active zone of the postsynaptic density domain (PSD) where neurotransmitter uptake takes place (Sclip et al., 2014). 3 - in the body, it governs apoptotic, necrotic and autophagic neuronal deaths, acting on different targets and intracellular organelles (Figure 1). More specifically, at the presynaptic terminal, JNK interacts with t-snare proteins, phosphorylating Syntaxin-1, Syntaxin-2, and Snap25-JNK, affecting the formation of the SNARE complex of synaptic vesicles and regulating vesicle closure and release. On the other hand, at the postsynaptic terminal JNK interacts with and phosphorylates the most abundant scaffolding protein in the PSD region PSD-95 and also Shank3, these scaffolds regulate N-methyl-D-aspartic acid and aminomethylphosphonic acid receptors in the PSD region (Kunde et al., 2013; Musi et al., 2022). In the body, JNK interacts with transcription factors, regulating c-Jun, activating transcription factors 2 and c-Fos in the nucleus, as well as with mitochondrial (among them Bcl2, Bclx, Beclin 1) and Golgi (repressing secretory traffic ). . . JNK has many physical targets such as caspases, Neuron H, SCG10, BAD, BIM family and many others. JNK is also involved in axonal transport (de Los Reyes Corrales et al., 2021), another important function in neurons. All of these physiological mechanisms are vital for a healthy brain, but they can be compromised, leading to brain disorders.

From this perspective, we examined JNK activation in Rett and Angelman syndromes and AD and tested whether its specific inhibition would confer neuronal protection. We selected two developmental disorders and one neurodegenerative disease (AD) as we searched for basic cellular and intracellular mechanisms and common key factors underlying synaptic dysfunction.

More specifically, in the context of Rett syndrome, a rare and severe developmental disorder characterized by pseudo-normal development, in which patients often reach normal neurodevelopmental milestones but begin to regress between 8 and 36 months of age, with loss of language and subtle disorders. motor coordination, motor dysfunction and manual stereotypy, previous work from our group investigated synaptic dysfunction in two different mouse models (Musi et al., 2021). We identified JNK as the major downstream player of CpG methylated protein 2 (MECP2), the gene mutated in the pathology. Both mouse models showed molecular disorganization of the PSD region in the postsynaptic element. Its disorganization has been associated with motor and cognitive impairments that characterize this disease. We show that specific inhibition of JNK by D-JNKI1, a cell-permeable peptide, strongly ameliorates symptoms and PSD domain disorganization in both mouse models. To understand the molecular mechanisms involved in the human disease, we used a patient-induced pluripotent stem cell (iPSC) model derived from Rett patient fibroblasts. We demonstrate JNK activation also in human neurons, differentiated from MECP2 mutant iPSCs, compared to the isogenic control expressing the wild-type MECP2 allele. JNK signaling was activated in MECP2 mutant iPSCs but not in control iPSCs, and D-JNKI1 blocked MECP2mut-induced neuronal death (Musi et al., 2021). Importantly, this is the first evidence that JNK is a key player in Human Rett syndrome.

In Angelman syndrome, another genetic developmental disorder characterized by autistic features, mental retardation, and motor impairments, we analyzed synaptic dysfunction in UBE3A^+/–mouse model, with UB3A being the mutated gene in this syndrome (Musi et al., 2020). JNK was also strongly activated in the brains of these mice, suggesting its important role in this neurodevelopmental disorder as well. In turn, these mice showed dysregulation of excitatory spine markers. D-JNKI1 treatment ameliorated their behavioral defects and this was associated with stabilization of synaptic biomarkers (Musi et al., 2020).

In neurodegenerative diseases, we mainly study AD, as JNK phosphorylates both amyloid precursor protein and Tau, the two key AD proteins, accelerating β-amyloid oligomer formation and neurofibrillary tangle deposition. JNK regulates the process of synaptic dysfunction inlivebut alsoin vitroAlzheimer's disease models. Indeed, in the animal model of AD, JNK inhibition strongly improved synaptic engagement, also improving cognitive performance (Sclip et al., 2014).

In summary, the data obtained from these studies strongly support the idea that JNK is a central player in the mechanisms of synaptic degeneration that characterize both neurodevelopmental and neurodegenerative diseases.Figure 1).

However, JNKs are a family (Jnk1, Jnk2 and Jnk3) of mitogen-activated protein kinases in which JNK1 and 2 are ubiquitous, while JNK3 is mainly expressed in neuronal tissue and is the most stress-sensitive isoform in the pathological context of the brain . , thus representing a more interesting and specific target. JNK3 plays an important role in a mouse model of AD (5×FAD mice) and, consequently, increased levels and activation of JNK3 were found in the postmortem brain of AD patients, but also in their cerebrospinal fluid (Gourmaud et al. . al. , 2015). These indications support the idea that JNK3 is a novel therapeutic target to combat AD and other brain diseases, which are strongly governed by synaptic dysfunction.

In the past, kinase inhibitors were considered dangerous, with non-specific activities and therefore very difficult to apply in clinical studies. However, thanks to the discovery of specific protein-protein interactions for the specific inhibition of kinases, the field is growing. Nowadays, in the field of drug discovery, kinases have become one of the most important targets in chronic and acute diseases. In fact, there are currently 68 FDA-approved drugs that target different protein kinases, six of which were approved last year.

Of note, specific inhibition of JNK3 can be used to modulate synaptic changes and prevent synaptic dysfunction in many different brain diseases, as shown by our research and the work of other groups. As brain damage and the underlying synaptic dysfunction continue to be the greatest burden on society, we strongly believe that it is vital to develop a strategy to protect synapses. We are now testing JNK3 inhibitors in new neurodegenerative models focusing on neuroinflammation to understand the potential of this particular inhibition to combat central nervous system dysfunctions.

We thank Prof. E. Welker for his advice and criticism and to Prof. M. Repici for reviewing the manuscript. We acknowledge the support of the University of Milan through the APC initiative. The MIUR Project Excellence grant and the UNIMI Research Support Plan, line 2 action C.

This work was funded by Finalized Research 2016 RF-2016-02361941, MIUR, -PON “Ricerca e Innovazione” PerMedNet id project ARS01-01226-RESEARCH PROJECTS OF RELEVANT NATIONAL INTEREST Prot. 2017MYJ5TH and 312 European Commission Horizon 2020 Research and Innovation Program No. 847749.

Editors C: Zhao M, Liu WJ, Wang Lu; Editor T: Jia Y

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