Loss of protein tyrosine phosphatase receptor delta PTPRD increases the number of cortical neurons, impairs synaptic function and induces autistic-like behaviors in adult mice.

BACKGROUND: The brain cortex is responsible for many higher-level cognitive functions. Disruptions during cortical development have long-lasting consequences on brain function and are associated with the etiology of brain disorders. We previously found that the protein tyrosine phosphatase receptor delta Ptprd, which is genetically associated with several human neurodevelopmental disorders, is essential to cortical brain development. Loss of Ptprd expression induced an aberrant increase of excitatory neurons in embryonic and neonatal mice by hyper-activating the pro-neurogenic receptors TrkB and PDGFRß in neural precursor cells. However, whether these alterations have long-lasting consequences in adulthood remains unknown. RESULTS: Here, we found that in Ptprd+/- or Ptprd-/- mice, the developmental increase of excitatory neurons persists through adulthood, affecting excitatory synaptic function in the medial prefrontal cortex. Likewise, heterozygosity or homozygosity for Ptprd also induced an increase of inhibitory cortical GABAergic neurons and impaired inhibitory synaptic transmission. Lastly, Ptprd+/- or Ptprd-/- mice displayed autistic-like behaviors and no learning and memory impairments or anxiety. CONCLUSIONS: These results indicate that loss of Ptprd has long-lasting effects on cortical neuron number and synaptic function that may aberrantly impact ASD-like behaviors.

Neural conditional ablation of the protein tyrosine phosphatase receptor Delta PTPRD impairs gliogenesis in the developing mouse brain cortex.

Neurodevelopmental disorders are characterized by alterations in the development of the cerebral cortex, including aberrant changes in the number and function of neural cells. Although neurogenesis is one of the most studied cellular processes in these pathologies, little evidence is known about glial development. Genetic association studies have identified several genes associated with neurodevelopmental disorders. Indeed, variations in the PTPRD gene have been associated with numerous brain disorders, including autism spectrum disorder, restless leg syndrome, and schizophrenia. We previously demonstrated that constitutive loss of PTPRD expression induces significant alterations in cortical neurogenesis, promoting an increase in intermediate progenitors and neurons in mice. However, its role in gliogenesis has not been evaluated. To assess this, we developed a conditional knockout mouse model lacking PTPRD expression in telencephalon cells. Here, we found that the lack of PTPRD in the mouse cortex reduces glial precursors, astrocytes, and oligodendrocytes. According to our results, this decrease in gliogenesis resulted from a reduced number of radial glia cells at gliogenesis onset and a lower gliogenic potential in cortical neural precursors due to less activation of the JAK/STAT pathway and reduced expression of gliogenic genes. Our study shows PTPRD as a regulator of the glial/neuronal balance during cortical neurodevelopment and highlights the importance of studying glial development to understand the etiology of neurodevelopmental diseases.

Correction: Vicencio et al. Transcriptional Signatures and Network-Based Approaches Identified Master Regulators Transcription Factors Involved in Experimental Periodontitis Pathogenesis. Int. J. Mol. Sci. 2023, 24, 14835.

In the original publication [...].

Transcriptional Signatures and Network-Based Approaches Identified Master Regulators Transcription Factors Involved in Experimental Periodontitis Pathogenesis.

Periodontitis is a chronic inflammatory disease characterized by the progressive and irreversible destruction of the periodontium. Its aetiopathogenesis lies in the constant challenge of the dysbiotic biofilm, which triggers a deregulated immune response responsible for the disease phenotype. Although the molecular mechanisms underlying periodontitis have been extensively studied, the regulatory mechanisms at the transcriptional level remain unclear. To generate transcriptomic data, we performed RNA shotgun sequencing of the oral mucosa of periodontitis-affected mice. Since genes are not expressed in isolation during pathological processes, we disclose here the complete repertoire of differentially expressed genes (DEG) and co-expressed modules to build Gene Regulatory Networks (GRNs) and identify the Master Transcriptional Regulators of periodontitis. The transcriptional changes revealed 366 protein-coding genes and 42 non-coding genes differentially expressed and enriched in the immune response. Furthermore, we found 13 co-expression modules with different representation degrees and gene expression levels. Our GRN comprises genes from 12 gene clusters, 166 nodes, of which 33 encode Transcription Factors, and 201 connections. Finally, using these strategies, 26 master regulators of periodontitis were identified. In conclusion, combining the transcriptomic analyses with the regulatory network construction represents a powerful and efficient strategy for identifying potential periodontitis-therapeutic targets.

c-Abl tyrosine kinase down-regulation as target for memory improvement in Alzheimer's disease.

Background: Growing evidence suggests that the non-receptor tyrosine kinase, c-Abl, plays a significant role in the pathogenesis of Alzheimer's disease (AD). Here, we analyzed the effect of c-Abl on the cognitive performance decline of APPSwe/PSEN1ΔE9 (APP/PS1) mouse model for AD. Methods: We used the conditional genetic ablation of c-Abl in the brain (c-Abl-KO) and pharmacological treatment with neurotinib, a novel allosteric c-Abl inhibitor with high brain penetrance, imbued in rodent's chow. Results: We found that APP/PS1/c-Abl-KO mice and APP/PS1 neurotinib-fed mice had improved performance in hippocampus-dependent tasks. In the object location and Barnes-maze tests, they recognized the displaced object and learned the location of the escape hole faster than APP/PS1 mice. Also, APP/PS1 neurotinib-fed mice required fewer trials to reach the learning criterion in the memory flexibility test. Accordingly, c-Abl absence and inhibition caused fewer amyloid plaques, reduced astrogliosis, and preserved neurons in the hippocampus. Discussion: Our results further validate c-Abl as a target for AD, and the neurotinib, a novel c-Abl inhibitor, as a suitable preclinical candidate for AD therapies.

Neuronal Rubicon Represses Extracellular APP/Amyloid ß Deposition in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.

The Autophagy Protein Pacer Positively Regulates the Therapeutic Potential of Mesenchymal Stem Cells in a Mouse Model of DSS-Induced Colitis.

Mesenchymal stem cells (MSC) have emerged as a promising tool to treat inflammatory diseases, such as inflammatory bowel disease (IBD), due to their immunoregulatory properties. Frequently, IBD is modeled in mice by using dextran sulfate sodium (DSS)-induced colitis. Recently, the modulation of autophagy in MSC has been suggested as a novel strategy to improve MSC-based immunotherapy. Hence, we investigated a possible role of Pacer, a novel autophagy enhancer, in regulating the immunosuppressive function of MSC in the context of DSS-induced colitis. We found that Pacer is upregulated upon stimulation with the pro-inflammatory cytokine TNFα, the main cytokine released in the inflammatory environment of IBD. By modulating Pacer expression in MSC, we found that Pacer plays an important role in regulating the autophagy pathway in this cell type in response to TNFα stimulation, as well as in regulating the immunosuppressive ability of MSC toward T-cell proliferation. Furthermore, increased expression of Pacer in MSC enhanced their ability to ameliorate the symptoms of DSS-induced colitis in mice. Our results support previous findings that autophagy regulates the therapeutic potential of MSC and suggest that the augmentation of autophagic capacity in MSC by increasing Pacer levels may have therapeutic implications for IBD.

LAR Receptor Tyrosine Phosphatase Family in Healthy and Diseased Brain.

Protein phosphatases are major regulators of signal transduction and they are involved in key cellular mechanisms such as proliferation, differentiation, and cell survival. Here we focus on one class of protein phosphatases, the type IIA Receptor-type Protein Tyrosine Phosphatases (RPTPs), or LAR-RPTP subfamily. In the last decade, LAR-RPTPs have been demonstrated to have great importance in neurobiology, from neurodevelopment to brain disorders. In vertebrates, the LAR-RPTP subfamily is composed of three members: PTPRF (LAR), PTPRD (PTPδ) and PTPRS (PTPσ), and all participate in several brain functions. In this review we describe the structure and proteolytic processing of the LAR-RPTP subfamily, their alternative splicing and enzymatic regulation. Also, we review the role of the LAR-RPTP subfamily in neural function such as dendrite and axon growth and guidance, synapse formation and differentiation, their participation in synaptic activity, and in brain development, discussing controversial findings and commenting on the most recent studies in the field. Finally, we discuss the clinical outcomes of LAR-RPTP mutations, which are associated with several brain disorders.

DEF8 and Autophagy-Associated Genes Are Altered in Mild Cognitive Impairment, Probable Alzheimer's Disease Patients, and a Transgenic Model of the Disease.

BACKGROUND: Disturbances in the autophagy/endolysosomal systems are proposed as early signatures of Alzheimer's disease (AD). However, few studies are available concerning autophagy gene expression in AD patients. OBJECTIVE: To explore the differential expression of classical genes involved in the autophagy pathway, among them a less characterized one, DEF8 (Differentially expressed in FDCP 8), initially considered a Rubicon family member, in peripheralblood mononuclear cells (PBMCs) from individuals with mild cognitive impairment (MCI) and probable AD (pAD) and correlate the results with the expression of DEF8 in the brain of 5xFAD mice. METHOD: By real-time PCR and flow cytometry, we evaluated autophagy genes levels in PBMCs from MCI and pAD patients. We evaluated DEF8 levels and its localization in brain samples of the 5xFAD mice by real-time PCR, western blot, and immunofluorescence. RESULTS: Transcriptional levels of DEF8 were significantly reduced in PBMCs of MCI and pAD patients compared with healthy donors, correlating with the MoCA and MoCA-MIS cognitive tests scores. DEF8 protein levels were increased in lymphocytes from MCI but not pAD, compared to controls. In the case of brain samples from 5xFAD mice, we observed a reduced mRNA expression and augmented protein levels in 5xFAD compared to age-matched wild-type mice. DEF8 presented a neuronal localization. CONCLUSION: DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially expressed in PBMCs of MCI and pAD and neurons of 5xFAD mice. These results suggest a potential role for DEF8 in the pathophysiology of AD.

Boldine inhibits the alveolar bone resorption during ligature-induced periodontitis by modulating the Th17/Treg imbalance.

BACKGROUND: During periodontitis, tooth-supporting alveolar bone is resorbed when there is an increased expression of the pro-osteolytic factor termed receptor activator of nuclear factor κB ligand (RANKL), which is responsible for osteoclast differentiation and activation. In periodontitis-affected tissues, the imbalance between T-helper type-17 (Th17) and T-regulatory (Treg) lymphocyte activity favors this RANKL overexpression. In this context, immunotherapeutic strategies aimed at modulating this Th17/Treg imbalance could eventually arrest the RANKL-mediated alveolar bone loss. Boldine has been reported to protect from pathological bone loss during rheumatoid arthritis and osteoporosis, whose pathogenesis is associated with a Th17/Treg imbalance. However, the effect of boldine on alveolar bone resorption during periodontitis has not been elucidated yet. This study aimed to determine whether boldine inhibits alveolar bone resorption by modulating the Th17/Treg imbalance during periodontitis. METHODS: Mice with ligature-induced periodontitis were orally treated with boldine (10/20/40 mg/kg) for 15 consecutive days. Non-treated periodontitis-affected mice and non-ligated mice were used as controls. Alveolar bone loss was analyzed by micro-computed tomography and scanning electron microscopy. Osteoclasts were quantified by histological identification of tartrate-resistant acid phosphatase-positive cells. Production of RANKL and its competitive antagonist osteoprotegerin (OPG) were analyzed by ELISA, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. The Th17 and Treg responses were analyzed by quantifying the T-cell frequency and number by flow cytometry. Also, the expression of their signature transcription factors and cytokines were quantified by qPCR. RESULTS: Boldine inhibited the alveolar bone resorption. Consistently, boldine caused a decrease in the osteoclast number and RANKL/OPG ratio in periodontal lesions. Besides, boldine reduced the Th17-lymphocyte detection and response and increased the Treg-lymphocyte detection and response in periodontitis-affected tissues. CONCLUSION: Boldine, administered orally, inhibited the alveolar bone resorption and modulated the Th17/Treg imbalance during experimental periodontitis.

Aggregatibacter Actinomycetemcomitans Induces Autophagy in Human Junctional Epithelium Keratinocytes.

The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.

IL-22-expressing CD4+ AhR+ T lymphocytes are associated with RANKL-mediated alveolar bone resorption during experimental periodontitis.

BACKGROUND AND OBJECTIVE: Over the past few years, the importance of interleukin-22 (IL-22) and T-helper (Th)22 lymphocytes in the pathogenesis of periodontitis has become apparent; however, there are still aspects that are not addressed yet. Cells expressing IL-22 and aryl hydrocarbon receptor (AhR), transcription factor master switch gene implicated in the differentiation and function of Th22 lymphocytes, have been detected in periodontal tissues of periodontitis-affected patients. In addition, IL-22 has been associated with osteoclast differentiation and their bone resorptive activity in vitro. However, the destructive potential of IL-22-expressing AhR+ Th22 lymphocytes over periodontal tissues during periodontitis has not been demonstrated in vivo yet. Therefore, this study aimed to analyze whether IL-22-expressing CD4+ AhR+ T lymphocytes detected in periodontal lesions are associated with alveolar bone resorption during experimental periodontitis. MATERIAL AND METHODS: Using a murine model of periodontitis, the expression levels of IL-22 and AhR, as well as the Th1-, Th2-, Th17- and T regulatory-associated cytokines, were analyzed in periodontal lesions using qPCR. The detection of CD4+ IL-22+ AhR+ T lymphocytes was analyzed in periodontal lesions and cervical lymph nodes that drain these periodontal lesions using flow cytometry. In addition, the expression of the osteoclastogenic mediator called receptor activator of nuclear factor-κB ligand (RANKL) was analyzed by qPCR, western blot, and immunohistochemistry. Finally, alveolar bone resorption was analyzed using micro-computed tomography and scanning electron microscopy, and the bone resorption levels were correlated with IL-22 and RANKL expression. RESULTS: Higher levels of IL-22, AhR, and RANKL, as well as IL-1ß, IL-6, IL-12, IL-17, IL-23, and TNF-α, were expressed in periodontal lesions of infected mice compared with periodontal tissues of sham-infected and non-infected controls. Similarly, high RANKL immunoreaction was observed in periodontal tissues of infected mice; however, few or absent RANKL immunoreaction was observed in controls. This association between RANKL and periodontal infection was ratified by western blot. Furthermore, a higher detection of CD4+ IL-22+ AhR+ T lymphocytes was found in periodontal lesions and cervical lymph nodes that drain these periodontal lesions in infected mice compared with non-infected controls. Finally, the increased IL-22 and RANKL expression showed positive correlation between them and with the augmented alveolar bone resorption observed in experimental periodontal lesions. CONCLUSION: This study demonstrates the increase of IL-22-expressing CD4+ AhR+ T lymphocytes in periodontitis-affected tissues and shows a positive correlation between IL-22, RANKL expression, and alveolar bone resorption.