A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g., lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value. However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals. Single-domain antibodies, called nanobodies, offer a range of possibilities toward therapy since they are easy to generate, express, produce, and modify, e.g., by linkage to nanobodies directed against other target molecules. Hence, we generated small MMP8-binding nanobodies, and established a proof-of-principle for developing nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.

Different animal models have been used to study the effects of prenatal protein undernutrition and the mechanisms by which these occur. In mammals, the maternal diet is manipulated, exerting both direct nutritional and indirect hormonal effects. Chicken embryos develop independent from the hen in the egg. Therefore, in the chicken, the direct effects of protein deficiency by albumen removal early during incubation can be examined. Prenatal protein undernutrition was established in layer-type eggs by the partial replacement of albumen by saline at embryonic day 1 (albumen-deprived group), compared to a mock-treated sham and a non-treated control group. At hatch, survival of the albumen-deprived group was lower compared to the control and sham group due to increased early mortality by the manipulation. No treatment differences in yolk-free body weight or yolk weight could be detected. The water content of the yolk was reduced, whereas the water content of the carcass was increased in the albumen-deprived group, compared to the control group, indicating less uptake of nutrients from the yolk. At embryonic day 16, 20 and at hatch, plasma triiodothyronine (T3), corticosterone, lactate or glucose concentrations and hepatic glycogen content were not affected by treatment. At embryonic day 20, the plasma thyroxine (T4) concentrations of the albumen-deprived embryos was reduced compared to the control group, indicating a decreased metabolic rate. Screening for differential protein expression in the liver at hatch using two-dimensional difference gel electrophoresis revealed not only changed abundance of proteins important for amino acid metabolism, but also of enzymes related to energy and glucose metabolism. Interestingly, GLUT1, a glucose transporter, and PCK2 and FBP1, two out of three regulatory enzymes of the gluconeogenesis were dysregulated. No parallel differences in gene expressions causing the differences in protein abundance could be detected pointing to post-transcriptional or post-translational regulation of the observed differences.

Monocular enucleation (ME) drastically affects the contralateral visual cortex, where plasticity phenomena drive specific adaptations to compensate for the unilateral loss of vision. In adult mice, complete reactivation of deprived visual cortex involves an early visually driven recovery followed by multimodal plasticity 3 to 7 weeks post ME (Van Brussel et al. [2011] Cereb. Cortex 21:2133-2146). Here, we specifically investigated the age dependence of the onset and the exact timing of both ME-induced reactivation processes by comparing cortical activity patterns of mice enucleated at postnatal day (P) 45, 90, or 120. A swifter open-eye potentiated reactivation characterized the binocular visual cortex of P45 mice. Nevertheless, even after 7 weeks, the reactivation remained incomplete, especially in the monocular cortex medial to V1. In comparison with P45, emergent cross-modal participation was demonstrated in P90 animals, although robust reactivation similar to enucleated adults (P120) was not achieved yet. Concomitantly, at 7 weeks post ME, somatosensory and auditory cortex shifted from a hypoactive state in P45 to hyperactivity in P120. Thus, we provide evidence for a presensitive period in which gradual recruitment of cross-modal recovery upon long-term ME coincides with the transition from adolescence to adulthood in mice.

Neurons in anterior cingulate cortex (aCC) project to dorsomedial striatum (DMS) as part of a corticostriatal circuit with putative roles in learning and other cognitive functions. In the present study, the spatial-cognitive importance of aCC and DMS was assessed in the hidden-platform version of the Morris water maze (MWM). Brain lesion experiments that focused on areas of connectivity between these regions indicated their involvement in spatial cognition. MWM learning curves were markedly delayed in DMS-lesioned mice in the absence of other major functional impairments, whereas there was a more subtle, but still significant influence of aCC lesions. Lesioned mice displayed impaired abilities to use spatial search strategies, increased thigmotaxic swimming, and decreased searching in the proximity of the escape platform. Additionally, aCC and DMS activity was compared in mice between the early acquisition phase (2 and 3 days of training) and the over-trained high-proficiency phase (after 30 days of training). Neuroplasticity-related expression of the immediate early gene Arc implicated both regions during the goal-directed, early phases of spatial learning. These results suggest the functional involvement of aCC and DMS in processes of spatial cognition that model associative cortex-dependent, human episodic memory abilities.

Worldwide, people are getting older, and this prolonged lifespan unfortunately also results in an increased prevalence of age-related neurodegenerative diseases, contributing to a diminished life quality of elderly. Age-associated neuropathies typically include diseases leading to dementia (Alzheimer's and Parkinson's disease), as well as eye diseases such as glaucoma and age-related macular degeneration. Despite many research attempts aiming to unravel aging processes and their involvement in neurodegeneration and functional decline, achieving healthy brain aging remains a challenge. The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived reported vertebrate that can be bred in captivity and displays many of the aging hallmarks that have been described for human aging, which makes it a very promising biogerontology model. As vision decline is an important hallmark of aging as well as a manifestation of many neurodegenerative diseases, we performed a comprehensive characterization of this fish's aging visual system. Our work reveals several aging hallmarks in the killifish retina and brain that eventually result in a diminished visual performance. Moreover, we found evidence for the occurrence of neurodegenerative events in the old killifish retina. Altogether, we introduce the visual system of the fast-aging killifish as a valuable model to understand the cellular and molecular mechanisms underlying aging in the vertebrate central nervous system. These findings put forward the killifish for target validation as well as drug discovery for rejuvenating or neuroprotective therapies ensuring healthy aging.

Aging increases the risk for neurodegenerative disease and brain trauma, both leading to irreversible and multifaceted deficits that impose a clear societal and economic burden onto the growing world population. Despite tremendous research efforts, there are still no treatments available that can fully restore brain function, which would imply neuroregeneration. In the adult mammalian brain, neuroregeneration is naturally limited, even more so in an aging context. In view of the significant influence of aging on (late-onset) neurological disease, it is a critical factor in future research. This review discusses the use of a non-standard gerontology model, the teleost brain, for studying the impact of aging on neurorepair. Teleost fish share a vertebrate physiology with mammals, including mammalian-like aging, but in contrast to mammals have a high capacity for regeneration. Moreover, access to large mutagenesis screens empowers these teleost species to fill the gap between established invertebrate and rodent models. As such, we here highlight opportunities to decode the factor age in relation to neurorepair, and we propose the use of teleost fish, and in particular killifish, to fuel new research in the neuro-gerontology field.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Cephalopods are emerging animal models and include iconic species for studying the link between genomic innovations and physiological and behavioral complexities. Coleoid cephalopods possess the largest nervous system among invertebrates, both for cell counts and brain-to-body ratio. Octopus vulgaris has been at the center of a long-standing tradition of research into diverse aspects of cephalopod biology, including behavioral and neural plasticity, learning and memory recall, regeneration, and sophisticated cognition. However, no chromosome-scale genome assembly was available for O. vulgaris to aid in functional studies. To fill this gap, we sequenced and assembled a chromosome-scale genome of the common octopus, O. vulgaris. The final assembly spans 2.8 billion basepairs, 99.34% of which are in 30 chromosome-scale scaffolds. Hi-C heatmaps support a karyotype of 1n = 30 chromosomes. Comparisons with other octopus species' genomes show a conserved octopus karyotype and a pattern of local genome rearrangements between species. This new chromosome-scale genome of O. vulgaris will further facilitate research in all aspects of cephalopod biology, including various forms of plasticity and the neural machinery underlying sophisticated cognition, as well as an understanding of cephalopod evolution.

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) in the eye, which ultimately results in visual impairment or even blindness. Because current therapies often fail to halt disease progression, there is an unmet need for novel neuroprotective therapies to support RGC survival. Various research lines suggest that visual target centers in the brain support RGC functioning and survival. Here, we explored whether increasing neuronal activity in one of these projection areas could improve survival of RGCs in a mouse glaucoma model. Prolonged activation of an important murine RGC target area, the superior colliculus (SC), was established via a novel optogenetic stimulation paradigm. By leveraging the unique channel kinetics of the stabilized step function opsin (SSFO), protracted stimulation of the SC was achieved with only a brief light pulse. SSFO-mediated collicular stimulation was confirmed by immunohistochemistry for the immediate-early gene c-Fos and behavioral tracking, which both demonstrated consistent neuronal activity upon repeated stimulation. Finally, the neuroprotective potential of optogenetic collicular stimulation was investigated in mice of either sex subjected to a glaucoma model and a 63% reduction in RGC loss was found. This work describes a new paradigm for optogenetic collicular stimulation and a first demonstration that increasing target neuron activity can increase survival of the projecting neurons.SIGNIFICANCE STATEMENT Despite glaucoma being a leading cause of blindness and visual impairment worldwide, no curative therapies exist. This study describes a novel paradigm to reduce retinal ganglion cell (RGC) degeneration underlying glaucoma. Building on previous observations that RGC survival is supported by the target neurons to which they project and using an innovative optogenetic approach, we increased neuronal activity in the mouse superior colliculus, a main projection target of rodent RGCs. This proved to be efficient in reducing RGC loss in a glaucoma model. Our findings establish a new optogenetic paradigm for target stimulation and encourage further exploration of the molecular signaling pathways mediating retrograde neuroprotective communication.

Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2(-/-) NK cells closely resembled that of Tbx21(-/-) NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21(-/-) NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells.

Unilateral vision loss through monocular enucleation (ME) results in partial reallocation of visual cortical territory to another sense in adult mice. The functional recovery of the visual cortex occurs through a combination of spared-eye potentiation and cross-modal reactivation driven by whisker-related, somatosensory inputs. Brain region-specific intracortical inhibition was recently recognized as a crucial regulator of the cross-modal component, yet the contribution of specific inhibitory neuron subpopulations remains poorly understood. Somatostatin (SST)-interneurons are ideally located within the cortical circuit to modulate sensory integration. Here we demonstrate that optogenetic stimulation of visual cortex SST-interneurons prior to eye removal decreases ME-induced cross-modal recovery at the stimulation site. Our results suggest that SST-interneurons act as local hubs, which are able to control the influx and extent of cortical cross-modal inputs into the deprived cortex. These insights critically expand our understanding of SST-interneuron-specific regulation of cortical plasticity induced by sensory loss.

GABAergic interneurons mainly originate in the medial ganglionic eminence (MGE) of the embryonic ventral telencephalon (VT) and migrate tangentially to the cortex, guided by membrane-bound and secreted factors. We found that Sip1 (Zfhx1b, Zeb2), a transcription factor enriched in migrating cortical interneurons, is required for their proper differentiation and correct guidance. The majority of Sip1 knockout interneurons fail to migrate to the neocortex and stall in the VT. RNA sequencing reveals that Sip1 knockout interneurons do not acquire a fully mature cortical interneuron identity and contain increased levels of the repulsive receptor Unc5b. Focal electroporation of Unc5b-encoding vectors in the MGE of wild-type brain slices disturbs migration to the neocortex, whereas reducing Unc5b levels in Sip1 knockout slices and brains rescues the migration defect. Our results reveal that Sip1, through tuning of Unc5b levels, is essential for cortical interneuron guidance.

Thin varicose centrifugal visual fibers, between 30-45 in number and displaying cGnRH-I immunoreactivity, were identified in Crocodylus niloticus. Approximately 80% of these fibers were also FMRF-amide-like immunoreactive. The cGnRH-I fibers extended from the preoptic region to the retina where they appeared to terminate in the external portion of the inner plexiform layer. The location of their neurons of origin could not be determined precisely following the intraocular injection of the retrograde axonal tracer RITC. Nevertheless, the presence of cGnRH-I-immunoreactive neurons exclusively within the complex comprising the terminal nerve and the septo-preoptic region, and of several retinopetal fibers labelled retrogradely with the axonal tracer at the septo-preoptic junction, indicates that the cGnRH-immunoreactive centrifugal visual system originates from within this complex.

This study compared the expression pattern, laminar distribution, and cell specificity of several rAAV serotypes (2/1, 2/5, 2/7, 2/8, and 2/9) injected in the primary visual cortex (V1) of adult C57Bl/6J mice. In order to obtain specific expression in certain neuron subtypes, different promoter sequences were evaluated for excitatory cell specificity: a universal cytomegalovirus (CMV) promoter, and two versions of the excitatory neuron-specific Ca(2+) /calmodulin-dependent kinase subunit α (CaMKIIα) promoter, CaMKIIα 0.4 and CaMKIIα 1.3. The spatial distribution as well as the cell type specificity was immunohistochemically verified. Depending on the rAAV serotype used, the transduced volume expressing reporter protein differed substantially (rAAV2/5 ≫ 2/7 ≈ 2/9 ≈ 2/8 ≫ 2/1). Excitatory neuron-specific targeting was promoter-dependent, with a surprising difference between the 1.3 kb and 0.4 kb CaMKIIα promoters. While CaMKIIα 1.3 and CMV carrying vectors were comparable, with 78% of the transduced neurons being excitatory for CMV and 82% for CaMKIIα 1.3, the shorter CaMKIIα 0.4 version resulted in 95% excitatory specificity. This study therefore puts forward the CaMKIIα 0.4 promoter as the best choice to target excitatory neurons with rAAVs. Together, these results can be used as an aid to select the most optimal vector system to deliver transgenes into specific rodent neocortical circuits, allowing further elucidation of their functions.

The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Cephalopods have evolved nervous systems that parallel the complexity of mammalian brains in terms of neuronal numbers and richness in behavioral output. How the cephalopod brain develops has only been described at the morphological level, and it remains unclear where the progenitor cells are located and what molecular factors drive neurogenesis. Using histological techniques, we located dividing cells, neural progenitors and postmitotic neurons in Octopus vulgaris embryos. Our results indicate that an important pool of progenitors, expressing the conserved bHLH transcription factors achaete-scute or neurogenin, is located outside the central brain cords in the lateral lips adjacent to the eyes, suggesting that newly formed neurons migrate into the cords. Lineage-tracing experiments then showed that progenitors, depending on their location in the lateral lips, generate neurons for the different lobes, similar to the squid Doryteuthis pealeii. The finding that octopus newborn neurons migrate over long distances is reminiscent of vertebrate neurogenesis and suggests it might be a fundamental strategy for large brain development.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR1A and 5-HTR3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR1A, 5-HTR2A and 5-HTR3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR1A and 5-HTR3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR2A and 5-HTR3A but not 5-HTR1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.

We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation.

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.