Cognitive aging is associated with redistribution of synaptic weights in the hippocampus.

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others' lifespans outlast their "mindspans."

Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-ß oligomers.

The cellular prion protein (PrPC) is a key neuronal receptor for ß-amyloid oligomers (AßO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AßO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AßO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AßO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AßO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AßO binding to PrPC Besides blocking AßO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aß and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AßO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

Extracellular Vesicles Isolated from Human Induced Pluripotent Stem Cell-Derived Neurons Contain a Transcriptional Network.

Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30-100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons.

Store depletion-induced h-channel plasticity rescues a channelopathy linked to Alzheimer's disease.

Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are misregulated in several neurological disorders, causing both gain- and loss-of-function channelopathies in neurons. Using several transgenic mouse models of Alzheimer's disease (AD), we find that sub-threshold voltage signals strongly influenced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels progressively deteriorate over chronological aging in hippocampal CA1 pyramidal neurons. The degraded signaling via HCN channels in the transgenic mice is accompanied by an age-related global loss of their non-uniform dendritic expression. Both the aberrant signaling via HCN channels and their mislocalization could be restored using a variety of pharmacological agents that target the endoplasmic reticulum (ER). Our rescue of the HCN channelopathy helps provide molecular details into the favorable outcomes of ER-targeting drugs on the pathogenesis and synaptic/cognitive deficits in AD mouse models, and implies that they might have beneficial effects on neurological disorders linked to HCN channelopathies.

Soluble Amyloid Precursor Protein α: Friend or Foe?

The "amyloidogenic" proteolytic processing of the cell surface amyloid precursor protein (APP) produces amyloid-ß, which causes a range of detrimental effects in the neuron, such as synaptic loss, and plays a key role in Alzheimer's disease. In contrast, "non-amyloidogenic" proteolytic processing, which involves the cleavage of APP by α-secretase, produces soluble amyloid precursor protein α (sAPPα) and is the most predominant proteolytic processing of APP in the healthy brain. Current research suggests that sAPPα plays a role in synaptic growth and plasticity, but whether this role is protective or detrimental is age-dependent. This review looks at the effects of increasing sAPPα during three time-points in life (in development, young adult, ageing/neurodegeneration) when synaptic plasticity plays an important role.

Inactivation of the microRNA-183/96/182 cluster results in syndromic retinal degeneration.

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

An NCAM mimetic, FGL, alters hippocampal cellular morphometry in young adult (4 month-old) rats.

The neural cell adhesion molecule, NCAM, is ubiquitously expressed within the CNS and has roles in development, cognition, neural plasticity and regulation of the immune system. NCAM is thus potentially an important pharmacological target for treatment of brain diseases. A cell adhesion mimetic FGL, a 15 amino-acid peptide derived from the second fibronectin type-III module of NCAM, has been shown to act as a neuroprotective agent in experimental disease and ageing models, restoring hippocampal/cognitive function and markedly alleviating deleterious changes in the CNS. However, the effects of FGL on the hippocampus of young healthy rats are unknown. The present study has examined the cellular neurobiological consequences of subcutaneous injections of FGL, on hippocampal cell morphometry in young (4 month-old) rats. We determined the effects of FGL on hippocampal volume, pyramidal neuron number/density (using unbiased quantitative stereology), and examined aspects of neurogenesis (using 2D morphometric analyses). FGL treatment reduced total volume of the dorsal hippocampus (associated with a decrease in total pyramidal neuron numbers in CA1 and CA3), and elevated the number of doublecortin immunolabeled neurons in the dentate gyrus, indicating a likely influence on neurogenesis in young healthy rats. These data indicate that FGL has a specific age dependent effect on the hippocampus, differing according to the development and maturity of the CNS.

3D models of neurodegeneration: implementation in drug discovery.

A lack of in vitro models that robustly represent the complex cellular pathologies underlying neurodegeneration has resulted in a translational gap between in vitro and in vivo results, creating a bottleneck in the development of new therapeutics. In the past decade, new and complex 3D models of the brain have been published at an exponential rate. However, many novel 3D models of neurodegeneration overlook the validation and throughput requirements for implementation in drug discovery. This therefore represents a knowledge gap that could hinder the translation of these models to drug discovery efforts. We review the recent progress in the development of 3D models of neurodegeneration, examining model design benefits and validation techniques, and discuss opportunities and standards for 3D models of neurodegeneration to be implemented in drug discovery and development.

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications.

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-ß deposition.

Alzheimer's disease (AD) is characterised by the aggregation and deposition of amyloid-ß (Aß) peptides in the human brain. In age-related late-onset AD, deficient degradation and clearance, rather than enhanced production, of Aß contributes to disease pathology. In the present study, we assessed the contribution of the two key Aß-degrading zinc metalloproteases, insulin-degrading enzyme (IDE) and neprilysin (NEP), to Aß degradation in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Using an Aß fluorescence polarisation assay, inhibition of IDE but not of NEP, blocked the degradation of Aß by human neurons. When the neurons were grown in a 3D extracellular matrix to visualise Aß deposition, inhibition of IDE but not NEP, increased the number of Aß deposits. The resulting Aß deposits were stained with the conformation-dependent, anti-amyloid antibodies A11 and OC that recognise Aß aggregates in the human AD brain. Inhibition of the Aß-forming ß-secretase prevented the formation of the IDE-inhibited Aß deposits. These data indicate that inhibition of IDE in live human neurons grown in a 3D matrix increased the deposition of Aß derived from the proteolytic cleavage of the amyloid precursor protein. This work has implications for strategies aimed at enhancing IDE activity to promote Aß degradation in AD.

Blended alginate/collagen hydrogels promote neurogenesis and neuronal maturation.

Brain extracellular matrix (ECM) is complex, heterogeneous and often poorly replicated in traditional 2D cell culture systems. The development of more physiologically relevant 3D cell models capable of emulating the native ECM is of paramount importance for the study of human induced pluripotent stem cell (iPSC)-derived neurons. Due to its structural similarity with hyaluronic acid, a primary component of brain ECM, alginate is a potential biomaterial for 3D cell culture systems. However, a lack of cell adhesion motifs within the chemical structure of alginate has limited its application in neural culture systems. This study presents a simple and accessible method of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation under physiological conditions and tests the hypothesis that such a substrate could influence the behaviour of human neurons in 3D culture. Regulation of the gelation process enabled the penetration of collagen fibrils throughout the hydrogel structure as demonstrated by transmission electron microscopy. Encapsulated human iPSC-derived neurons adhered to the blended hydrogel as evidenced by the increased expression of α1, α2 and ß1 integrins. Furthermore, immunofluorescence microscopy revealed that encapsulated neurons formed complex neural networks and matured into branched neurons expressing synaptophysin, a key protein involved in neurotransmission, along the neurites. Mechanical tuning of the hydrogel stiffness by modulation of the alginate ionic crosslinker concentration also influenced neuron-specific gene expression. In conclusion, we have shown that by tuning the physicochemical properties of the alginate/collagen blend it is possible to create different ECM-like microenvironments where complex mechanisms underpinning the growth and development of human neurons can be simulated and systematically investigated.

WITHDRAWN: Dendritic Spine and Synapse Morphological Alterations Induced by a Neural Cell Adhesion Molecule (NCAM) Mimetic.

The neural cell adhesion molecule (NCAM) is a glycoprotein expressed on the surface of neurons and glial cells. It plays a key role in morphogenesis of the nervous system, regeneration of damaged neural tissue and synaptic plasticity. The extracellular domain of NCAM engages in homophilic interactions (NCAM binding to NCAM) and in heterophilic interactions between NCAM and other proteins such as the fibroblast growth factor (FGF) receptor. It promotes synaptogenesis and activity-dependent remodelling of synapses but less is know of its influence on synaptic and dendritic morphology. Recently, quantitative electron microscopy and 3-dimensional reconstruction (3-D) of ultrathin serial sections has been used to examine the morphology of synapses and dendritic spines in the hippocampus of rats treated with a neural cell adhesion molecule-derived fibroblast growth factor receptor agonist, FGL-peptide (an NCAM mimetic). These data show clearly that the FGL peptide has marked influences on both spine and synaptic form.

Tau Proteolysis in the Pathogenesis of Tauopathies: Neurotoxic Fragments and Novel Biomarkers.

With predictions showing that 131.5 million people worldwide will be living with dementia by 2050, an understanding of the molecular mechanisms underpinning disease is crucial in the hunt for novel therapeutics and for biomarkers to detect disease early and/or monitor disease progression. The metabolism of the microtubule-associated protein tau is altered in different dementias, the so-called tauopathies. Tau detaches from microtubules, aggregates into oligomers and neurofibrillary tangles, which can be secreted from neurons, and spreads through the brain during disease progression. Post-translational modifications exacerbate the production of both oligomeric and soluble forms of tau, with proteolysis by a range of different proteases being a crucial driver. However, the impact of tau proteolysis on disease progression has been overlooked until recently. Studies have highlighted that proteolytic fragments of tau can drive neurodegeneration in a fragment-dependent manner as a result of aggregation and/or transcellular propagation. Proteolytic fragments of tau have been found in the cerebrospinal fluid and plasma of patients with different tauopathies, providing an opportunity to develop these fragments as novel disease progression biomarkers. A range of therapeutic strategies have been proposed to halt the toxicity associated with proteolysis, including reducing protease expression and/or activity, selectively inhibiting protease-substrate interactions, and blocking the action of the resulting fragments. This review highlights the importance of tau proteolysis in the pathogenesis of tauopathies, identifies putative sites during tau fragment-mediated neurodegeneration that could be targeted therapeutically, and discusses the potential use of proteolytic fragments of tau as biomarkers for different tauopathies.

Identification of TMEM230 mutations in familial Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson's disease or parkinsonian disorders. The pathogenesis of Parkinson's disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body-confirmed Parkinson's disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson's disease, with implications for understanding the pathogenic mechanism of Parkinson's disease and for developing rational therapies.

Coupling of human nicotinic acetylcholine receptors alpha 7 to calcium channels in GH3 cells.

The neuronal nicotinic acetylcholine receptor alpha7 (nAChR alpha7) may be involved in cognitive deficits in Schizophrenia and Alzheimer's disease. A fast pharmacological characterization of homomeric alpha7 receptors is mostly hampered by their low functional expression levels in heterologous expression systems. In the present study expression of homomeric nAChR alpha7 was achieved in GH3 rat pituitary cells. Alpha7 subunits were heterologously expressed as components of [125I]-labeled alpha-bungarotoxin binding nAChRs (Bmax: 1.2 pmol/mg protein). Function of the expressed alpha7 ion channels was assessed by patch-clamp recording and calcium imaging. While acetylcholine-induced currents desensitized within much less than 1 s, calcium-sensitive fluorescence transients peaked after 5-10 s and returned to background levels within 30 s only. The fluorescence signal was blocked by isradipine and removal of extracellular sodium indicated that in these cells opening of rapidly desensitizing alpha7 nAChR triggers calcium influx via voltage-gated, DHP-sensitive calcium channels. In this cellular system, agonists revealed the following rank order of potency: epibatidine>anatoxin A>AAR17779>ABT-594>DMPP>nicotine>GTS-21>cytisine>ABT-418>acetylcholine>choline>ABT-089. All of the signals were inhibited by the alpha7 antagonists alpha-bungarotoxin (pIC50: 7.4) and methyllycaconitine (pIC50: 7.8). Further, marketed antidepressants showed antagonistic activity with the following rank order of potency: fluoxetine>imipramine>paroxetine>sertraline. These data illustrate that coupling to voltage-gated calcium channels allows a rapid and reliable functional examination of nAChR alpha7.

Amyloid-beta induced CA1 pyramidal cell loss in young adult rats is alleviated by systemic treatment with FGL, a neural cell adhesion molecule-derived mimetic peptide.

Increased levels of neurotoxic amyloid-beta in the brain are a prominent feature of Alzheimer's disease. FG-Loop (FGL), a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 µl amyloid-beta(25-35) injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-beta(25-35) injection. NeuN, a neuronal marker (for nuclear staining) was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3beta (GSK3ß) and to determine the effects of amyloid-beta(25-35) and FGL on the activation state of GSK3ß, since active GSK3ß has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-beta caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-beta was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3ß, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3ß after FGL treatment. These data suggest that FGL, although potentially disruptive in non-pathological conditions, can be neuroprotective in disease-like conditions.