Interneuron Simplification and Loss of Structural Plasticity As Markers of Aging-Related Functional Decline.

Changes in excitatory neuron and synapse structure have been recognized as a potential physical source of age-related cognitive decline. Despite the importance of inhibition to brain plasticity, little is known regarding aging-associated changes to inhibitory neurons. Here we test for age-related cellular and circuit changes to inhibitory neurons of mouse visual cortex. We find no substantial difference in inhibitory neuron number, inhibitory neuronal subtypes, or synapse numbers within the cerebral cortex of aged mice compared with younger adults. However, when comparing cortical interneuron morphological parameters, we find differences in complexity, suggesting that arbors are simplified in aged mice. In vivo two-photon microscopy has previously shown that in contrast to pyramidal neurons, inhibitory interneurons retain a capacity for dendritic remodeling in the adult. We find that this capacity diminishes with age and is accompanied by a shift in dynamics from balanced branch additions and retractions to progressive prevalence of retractions, culminating in a dendritic arbor that is both simpler and more stable. Recording of visually evoked potentials shows that aging-related interneuron dendritic arbor simplification and reduced dynamics go hand in hand with loss of induced stimulus-selective response potentiation (SRP), a paradigm for adult visual cortical plasticity. Chronic treatment with the antidepressant fluoxetine reversed deficits in interneuron structural dynamics and restored SRP in aged animals. Our results support a structural basis for age-related impairments in sensory perception, and suggest that declines in inhibitory neuron structural plasticity during aging contribute to reduced functional plasticity.SIGNIFICANCE STATEMENT Structural alterations in neuronal morphology and synaptic connections have been proposed as a potential physical basis for age-related decline in cognitive function. Little is known regarding aging-associated changes to inhibitory neurons, despite the importance of inhibitory circuitry to adult cortical plasticity and the reorganization of cortical maps. Here we show that brain aging goes hand in hand with progressive structural simplification and reduced plasticity of inhibitory neurons, and a parallel decline in sensory map plasticity. Fluoxetine treatment can attenuate the concurrent age-related declines in interneuron structural and functional plasticity, suggesting it could provide an important therapeutic approach for mitigating sensory and cognitive deficits associated with aging.

CPG15 regulates synapse stability in the developing and adult brain.

Use-dependent selection of optimal connections is a key feature of neural circuit development and, in the mature brain, underlies functional adaptation, such as is required for learning and memory. Activity patterns guide circuit refinement through selective stabilization or elimination of specific neuronal branches and synapses. The molecular signals that mediate activity-dependent synapse and arbor stabilization and maintenance remain elusive. We report that knockout of the activity-regulated gene cpg15 in mice delays developmental maturation of axonal and dendritic arbors visualized by anterograde tracing and diolistic labeling, respectively. Electrophysiology shows that synaptic maturation is also delayed, and electron microscopy confirms that many dendritic spines initially lack functional synaptic contacts. While circuits eventually develop, in vivo imaging reveals that spine maintenance is compromised in the adult, leading to a gradual attrition in spine numbers. Loss of cpg15 also results in poor learning. cpg15 knockout mice require more trails to learn, but once they learn, memories are retained. Our findings suggest that CPG15 acts to stabilize active synapses on dendritic spines, resulting in selective spine and arbor stabilization and synaptic maturation, and that synapse stabilization mediated by CPG15 is critical for efficient learning.

Reversible modulations of neuronal plasticity by VEGF.

Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.

VEGF is required for dendritogenesis of newly born olfactory bulb interneurons.

The angiogenic factor vascular endothelial growth factor A (VEGF) has been shown to have a role in neurogenesis, but how it affects adult neurogenesis is not fully understood. To delineate a role for VEGF in successive stages of olfactory bulb (OB) neurogenesis, we used a conditional transgenic system to suppress VEGF signaling at the adult mouse sub-ventricular zone (SVZ), rostral migratory stream (RMS) and OB, which constitute the respective sites of birth, the migration route, and sites where newly born interneurons mature and integrate within the existing OB circuitry. Following the development of fluorescently tagged adult-born neurons, we show that sequestration of VEGF that is constitutively expressed by distinct types of resident OB neurons greatly impaired dendrite development in incoming SVZ-born neurons. This was evidenced by reduced dendritic spine density of granule cells and significantly shorter and less branched dendrites in periglomerular neurons. Notably, the vasculature and perfusion of the SVZ, RMS and OB were not adversely affected when VEGF suppression was delayed until after birth, thus uncoupling the effect of VEGF on dendritogenesis from its known role in vascular maintenance. Furthermore, a requirement for VEGF was specific to newly born neurons, as already established OB neurons were not damaged by VEGF inhibition. This study thus uncovered a surprising perfusion-independent role of VEGF in the adult brain, namely, an essential role in the maturation of adult-born neurons.

High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with next generation sequencing.

The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.

A novel approach for enzyme replacement therapy. The use of phenylalanine hydroxylase-based fusion proteins for the treatment of phenylketonuria.

Metabolic diseases arise from mutations in key enzymes of major metabolic pathways. One promising approach for the treatment of such diseases is based on the administration of a wild-type enzyme to substitute the activity of the impaired enzyme by the use of enzyme replacement therapy, yet it is important to deliver this enzyme to the specific deficient tissue. We suggest a new concept for the treatment of metabolic diseases using fusion proteins. We examined the feasibility of this concept in the well characterized metabolic disease, phenylketonuria (PKU), which results from a mutation in the liver enzyme phenylalanine hydroxylase (PAH). PAH is a key enzyme in the metabolic pathway of phenylalanine. Deficiency in PAH leads to high and persistent levels of this amino acid in the plasma of PKU patients, causing permanent neurological damage. Currently a low protein diet is still considered the only effective treatment for most PKU patients. To restore PAH activity in the liver of PKU patients, we constructed PAH-based fusion proteins with delivery moieties based on the HIV-transactivator of transcription peptide, and fragments of human hepatocyte growth factor aiming to specifically target PAH to the liver. We show that these new fusion proteins can be delivered into a variety of human liver cell lines and retain PAH activity after being internalized. We also show that plasma phenylalanine levels were dramatically lowered in mice treated with PAH-based fusion proteins after intravenous administration. We therefore suggest an alternative concept for the treatment of PKU using targeted fusion proteins, which may also be applied to the treatment of other metabolic diseases.