Retinitis pigmentosa (RP) is an inherited retinopathy that leads to photoreceptor loss. RP has been related to oxidative stress, autophagy, and inflammation. This study aimed to identify changes in the levels of oxidative stress and autophagy markers in the retina of control and rd10 mice during different phases of retinal development. Changes in the retinal oxidation system were investigated by measuring the levels of oxidized and reduced glutathione (GSH/GSSG), retinal avidin-positive cells, and 4-hydroxynonenal (4-HNE) staining intensity. Autophagy characterization was explored by measuring the levels of microtubule-associated protein 1 light chain 3 (LC3), beclin, autophagy-related proteins 5 and 7 (Atg5 and Atg7), and lysosomal associated membrane protein-2A (LAMP-2A). At P28 retinal GSH concentrations decreased in rd10 mice compared to the controls. No differences were found in retinal GSSG concentrations between the control and rd10 mice. There was an increase in retinal GSSG concentrations and a decrease in the GSH/GSSG ratio in the control and rd10 mice at P21 and P28 compared to P13. We observed an increase in avidin-positive cells in rd10 retinas. 4-HNE was increased in rd10 retinas at P13, and it also increased in control mice with age. We did not observe any differences in the retinal levels of LC3II/I ratio, Beclin, Atg5, or Atg7 in the rd10 mice compared to the controls. There was an increase in the LAMP-2A concentrations in the control and rd10 mice with development age (P28 concentrations vs. P13). Although only slight differences were found in the oxidative stress and autophagy markers between the control and rd10 mice, there were increases in the GSSG, 4-HNE, and LAMP-2A with age. This increase in the oxidative stress and chaperone-mediated autophagy has not been described before and occurred just after the mice opened their eyes, potentially indicating a retinal response to light exposure.

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.

Objectives: Mammary tumours in cats are biologically aggressive. The standard of care relies upon wide surgical resection. Chemotherapy has been described in the macroscopic disease setting; however, limited efficacy has been shown. The aim of this study was to assess the efficacy of toceranib phosphate in macroscopic feline mammary tumours (FMTs). Methods: A total of 17 cats with cytologically or histopathologically confirmed mammary adenocarcinoma (gross disease) were prospectively enrolled. Toceranib phosphate was administered at a median dose of 2.77 mg/kg (range 2.3–3.2) PO q48 h. No corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) were administered. Toxicity was graded according to Veterinary Cooperative Oncology Group–Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 criteria. The response was assessed after 1 month, following Response Evaluation Criteria In Solid Tumours (RECIST) criteria. Results: Toxicity was observed in eight cats, with most instances being grade 1 or 2, which were managed with supportive care. Only one cat experienced grade 3 toxicity (anorexia), which resolved after a dose reduction. Clinical benefit was seen in 12 (64.7%) cats and an objective response was seen in six (35.2%) cats. One cat experienced complete response, five had partial response, six had stable disease and five had progressive disease. One cat showed distant progression (malignant pleural effusion) despite continued partial remission of the primary tumour. The median progression-free survival and median overall survival time were 91 days (range 30–158) and 145 days (range 31–234), respectively. Conclusions and relevance: Toceranib phosphate showed clinical benefit and a good safety profile in advanced or recurrent FMTs, offering a new alternative in the treatment of this disease; however, further prospective and randomised studies are required to further assess its efficacy. Interestingly, one cat developed distant metastases while the primary tumour showed partial response, suggesting that primary tumour and metastatic disease may not sustain the same sensitivity to toceranib.

In the subventricular zone (SVZ), neural stem cells (NSCs) generate neural progenitor cells (NPCs), which proliferate and differentiate into neuroblasts (NBs) that will travel along the rostral migratory stream (RMS) to the olfactory bulbs (OBs), where they mature into interneurons. Rnd3, a member of the Rho GTPase family, regulates cytoskeletal dynamics, neuronal morphology, and survival, primarily by interacting with the RhoA/ROCK pathway. In the central nervous system, Rnd3 is highly expressed during early postnatal development and is essential for neural function, axonal myelination, and neuronal polarization, as its deficiency leads to severe motor and neurodevelopmental impairments. In this study we show that NBs from Rnd3 KO mice accumulate in the SVZ and that these are principally characterized as late/migrating NBs. We investigated whether the observed accumulation results from increased proliferation and/or differentiation potential of NSCs and NPCs, and/or altered NB migration to the OBs through the RMS, potentially accompanied by increased proliferation. Our in vitro experiments indicate that the loss of Rnd3 does not affect NSC behavior. In addition, RNA sequencing reveals that Rnd3 expression is highest in NBs, particularly in late-stage NBs, suggesting a potential role in migration. Furthermore, gene expression analyses indicate that the loss of Rnd3 may disrupt NB cytoskeletal dynamics by altering the expression of key components of the RhoA/ROCK signaling pathway. These findings provide mechanistic insights into how Rnd3 deletion impairs NB migration.

Background: Multiple myeloma (MM) is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to resistance to treatment. The CRISPR/Cas9 system has emerged as a powerful tool for understanding the role of different genetic alterations in the pathogenesis of hematologic malignancies in both cell lines and mouse models. Despite current advances of gene editing tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy. Results: We have designed different guide RNAs and cloning them into a lentiviral plasmid, which contains all the machinery necessary for developing the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids to produce lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Additionally, the impact of Rnd3 expression absence was analyzed by a transcriptomic analysis consisting of 3’ UTR RNA sequencing. The Rnd3 knock-down cells showed a different transcriptomic profile in comparison to control cells. Conclusions: We have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines, and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines. Furthermore, Rnd3 silencing produces changes in the cellular transcriptomic profile.

Canine inflammatory mammary carcinoma (IMC) is an aggressive and rare type of mammary gland cancer in dogs where vascular endothelial growth factor and cyclooxigenase-2 overexpression usually occur, which contribute to its invasive and angiogenic nature. This study aimed to evaluate the efficacy and safety of a combined treatment regimen of toceranib phosphate and carprofen in dogs with measurable IMC. Fifteen female dogs with histopathologically confirmed IMC were included, undergoing a regimen of toceranib (2.4–2.75 mg/kg PO, three times weekly) and carprofen (4.4 mg/kg/24 h PO). Initial evaluations included physical exams, tumor measurements, complete blood count, biochemistry, urinalysis, three view thoracic radiographs, and abdominal ultrasound. Follow-up assessments of physical condition and quality of life (QOL) were conducted bi-weekly, with tumor response evaluations monthly, using RECIST v1.0 criteria. While no complete or partial responses were observed, 60% of the dogs maintained stable disease, with a median progression-free survival of 76 days and an overall survival of 90 days. Notably, 60% of the dogs showed clinical benefit through improved QOL and disease stabilization. The treatment was well-tolerated, with only grade I/II toxicities reported. Despite limited biological activity against the cancer, this protocol may enhance QOL in dogs with IMC, offering a valuable palliative option.