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Epidemiological studies have shown a clear association between early life zinc deficiency and Autism Spectrum Disorders (ASD). In line with this, mouse models have revealed prenatal zinc deficiency as a profound risk factor for neurobiological and behavioral abnormalities in the offspring reminiscent of ASD behavior. From these studies, a complex pathology emerges, with alterations in the gastrointestinal and immune system and synaptic signaling in the brain, as a major consequence of prenatal zinc deficiency. The features represent a critical link in a causal chain that leads to various neuronal dysfunctions and behavioral phenotypes observed in prenatal zinc deficient (PZD) mice and probably other mouse models for ASD. Given that the complete phenotype of PZD mice may be key to understanding how non-genetic factors can modify the clinical features and severity of autistic patients and explain the observed heterogeneity, here, we summarize published data on PZD mice. We critically review the emerging evidence that prenatal zinc deficiency is at the core of several environmental risk factors associated with ASD, being mechanistically linked to ASD-associated genetic factors. In addition, we highlight future directions and outstanding questions, including potential symptomatic, disease-modifying, and preventive treatment strategies.

Autism Spectrum Disorders (ASD) are caused by a combination of genetic predisposition and nongenetic factors. Among the nongenetic factors, maternal immune system activation and zinc deficiency have been proposed. Intriguingly, as a genetic factor, copy-number variations in S100B, a pro-inflammatory damage-associated molecular pattern (DAMP), have been associated with ASD, and increased serum S100B has been found in ASD. Interestingly, it has been shown that increased S100B levels affect zinc homeostasis in vitro. Thus, here, we investigated the influence of increased S100B levels in vitro and in vivo during pregnancy in mice regarding zinc availability, the zinc-sensitive SHANK protein networks associated with ASD, and behavioral outcomes. We observed that S100B affects the synaptic SHANK2 and SHANK3 levels in a zinc-dependent manner, especially early in neuronal development. Animals exposed to high S100B levels in utero similarly show reduced levels of free zinc and SHANK2 in the brain. On the behavioral level, these mice display hyperactivity, increased stereotypic and abnormal social behaviors, and cognitive impairment. Pro-inflammatory factors and zinc-signaling alterations converge on the synaptic level revealing a common pathomechanism that may mechanistically explain a large share of ASD cases., (© 2021. The Author(s).)

In the last years, research has shown that zinc ions play an essential role in the physiology of brain function. Zinc acts as a potent neuromodulatory agent and signaling ions, regulating healthy brain development and the function of both neurons and glial cells. Therefore, the concentration of zinc within the brain and its cells is tightly controlled. Zinc transporters are key regulators of (extra-) cellular zinc levels, and deregulation of zinc homeostasis and zinc transporters has been associated with neurodegenerative and neuropsychiatric disorders. However, to date, the presence of specific family members and their subcellular localization within brain cells have not been investigated in detail. Here, we analyzed the expression of all zinc transporters (ZnTs) and Irt-like proteins (ZIPs) in the rat brain. We further used primary rat neurons and rat astrocyte cell lines to differentiate between the expression found in neurons or astrocytes or both. We identified ZIP4 expressed in astrocytes but significantly more so in neurons, a finding that has not been reported previously. In neurons, ZIP4 is localized to synapses and found in a complex with major postsynaptic scaffold proteins of excitatory synapses. Synaptic ZIP4 reacts to short-term fluctuations in local zinc levels. We conclude that ZIP4 may have a so-far undescribed functional role at excitatory postsynapses.

The accumulation of amyloid β (Aβ) triggers a cascade of toxic events in Alzheimer's disease (AD). The KLVFF peptide can interfere with Aβ aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood-brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aβ-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD., Competing Interests: The authors declare no conflict of interest.

Increasing evidence links proteins of the S100 family to the pathogenesis of Alzheimer's disease (AD). S100 proteins are EF-hand calcium-binding proteins with intra- and extracellular functions related to regulation of proliferation, differentiation, apoptosis, and trace metal homeostasis, and are important modulators of inflammatory responses. For example, S100A6, S100A8, and S100B expression levels were found increased in inflammatory diseases, but also neurodegenerative disorders, and S100A8/A9 complexes may provide a mechanistic link between amyloid-beta (Aβ) plaque formation and neuroinflammation. On the other hand, S100B, a proinflammatory protein that is chronically up-regulated in AD and whose elevation precedes plaque formation, was recently shown to suppress Aβ aggregation. Here, we report expression of S100A6 and S100B in astrocytes and less so in neurons, and low level of expression of S100A8 in both neurons and glial cells in vitro . In vivo , S100A8 expression is almost absent in the brain of aged wildtype mice, while S100A6 and S100B are expressed in all brain regions and most prominently in the cortex and cerebellum. S100B seems to be enriched in Purkinje cells of the cerebellum. In contrast, in the brain of APP23 mice, a mouse model for Alzheimer's disease, S100B, S100A6, and S100A8 show co-localization with Aβ plaques, compatible with astrocyte activation, and the expression level of S100A8 is increased in neural cells. While S100A6 and S100B are enriched in the periphery of plaques where less fibrillar Aβ is found, S100A8 is more intense within the center of the inclusion. In vitro assays show that, similarly to S100B, S100A6, and S100A8 also delay Aβ aggregation suggesting a regulatory action over protein aggregation. We posit that elevated expression levels and overlapping spatial distribution of brain S100 proteins and plaques translates functional relationships between these inflammatory mediators and AD pathophysiology processes that uncover important molecular mechanisms linking the aggregation and neuroinflammation cascades.

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer's disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aβ), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aβ degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The loss of one copy of SHANK3 (SH3 and multiple ankyrin repeat domains 3) in humans highly contributes to Phelan McDermid syndrome (PMDS). In addition, SHANK3 was identified as a major autism candidate gene. Interestingly, the protein encoded by the SHANK3 gene is regulated by zinc. While zinc deficiency depletes synaptic pools of Shank3, increased zinc levels were shown to promote synaptic scaffold formation. Therefore, the hypothesis arises that patients with PMDS and Autism caused by Shankopathies, having one intact copy of SHANK3 left, may benefit from zinc supplementation, as elevated zinc may drive remaining Shank3 into the post-synaptic density (PSD) and may additional recruit Shank2, a second zinc-dependent member of the SHANK gene family. Further, elevated synaptic zinc levels may modulate E/I ratios affecting other synaptic components such as NMDARs. However, several factors need to be considered in relation to zinc supplementation such as the role of Shank3 in the gastrointestinal (GI) system-the location of zinc absorption in humans. Therefore, here, we briefly discuss the prospect and impediments of zinc supplementation in disorders affecting Shank3 such as PMDS and propose a model for most efficacious supplementation.

Neuronal metal ions such as zinc are essential for brain function. In particular synaptic processes are tightly related to metal and protein homeostasis, for example through extracellular metal-binding proteins. One such protein is neuronal S100B, a calcium and zinc binding damage-associated molecular pattern (DAMP), whose chronic upregulation is associated with aging, Alzheimer's disease (AD), motor neuron disease and traumatic brain injury (TBI). Despite gained insights on the structure of S100B, it remains unclear how its calcium and zinc binding properties regulate its function on cellular level. Here we report a novel role of S100B in trace metal homeostasis, in particular the regulation of zinc levels in the brain. Our results show that S100B at increased extracellular levels is not toxic, persists at high levels, and is taken up into neurons, as shown by cell culture and biochemical analysis. Combining protein bioimaging and zinc quantitation, along with a zinc-binding impaired S100B variant, we conclude that S100B effectively scavenges zinc ions through specific binding, resulting in a redistribution of the intracellular zinc pool. Our results indicate that scavenging of zinc by increased levels of S100B affects calcium levels in vitro . Thereby S100B is able to mediate the cross talk between calcium and zinc homeostasis. Further, we investigated a possible new neuro-protective role of S100B in excitotoxicity via its effects on calcium and zinc homeostasis. Exposure of cells to zinc-S100B but not the zinc-binding impaired S100B results in an inhibition of excitotoxicity. We conclude that in addition to its known functions, S100B acts as sensor and regulator of elevated zinc levels in the brain and this metal-buffering activity is tied to a neuroprotective role.

Zn is essential for growth and development. The bioavailability of Zn is affected by several factors such as other food components. It is therefore of interest, to understand uptake mechanisms of Zn delivering compounds to identify ways to bypass the inhibitory effects of these factors. Here, we studied the effect of Zn amino acid conjugates (ZnAAs) on the bioavailabilty of Zn. We used Caco-2 cells and enterocytes differentiated from human induced pluripotent stem cells from a control and Acrodermatitis enteropathica (AE) patient, and performed fluorescence based assays, protein biochemistry and atomic absorption spectrometry to characterize cellular uptake and absorption of ZnAAs. The results show that ZnAAs are taken up by AA transporters, leading to an intracellular enrichment of Zn mostly uninhibited by Zn uptake antagonists. Enterocytes from AE patients were unable to gain significant Zn through exposure to ZnCl 2 but did not show differences with respect to ZnAAs. We conclude that ZnAAs may possess an advantage over classical Zn supplements such as Zn salts, as they may be able to increase bioavailability of Zn, and may be more efficient in patients with AE.

Phelan McDermid Syndrome (PMDS) is a genetic disorder characterized by features of Autism spectrum disorders. Similar to reports of Zn deficiency in autistic children, we have previously reported high incidence of Zn deficiency in PMDS. However, the underlying mechanisms are currently not well understood. Here, using inductively coupled plasma mass-spectrometry to measure the concentration of Zinc (Zn) and Copper (Cu) in hair samples from individuals with PMDS with 22q13.3 deletion including SHANK3 (SH3 and multiple ankyrin repeat domains 3), we report a high rate of abnormally low Zn/Cu ratios. To investigate possible underlying mechanisms, we generated enterocytes from PMDS patient-derived induced pluripotent stem cells and used Caco-2 cells with knockdown of SHANK3. We detected decreased expression of Zn uptake transporters ZIP2 and ZIP4 on mRNA and protein level correlating with SHANK3 expression levels, and found reduced levels of ZIP4 protein co-localizing with SHANK3 at the plasma membrane. We demonstrated that especially ZIP4 exists in a complex with SHANK3. Furthermore, we performed immunohistochemistry on gut sections from Shank3αβ knockout mice and confirmed a link between enterocytic SHANK3, ZIP2 and ZIP4. We conclude that apart from its well-known role in the CNS, SHANK3 might play a specific role in the GI tract.

Apart from teratogenic and pathological effects of zinc deficiency such as the occurrence of skin lesions, anorexia, growth retardation, depressed wound healing, altered immune function, impaired night vision, and alterations in taste and smell acuity, characteristic behavioral changes in animal models and human patients suffering from zinc deficiency have been observed. Given that it is estimated that about 17% of the worldwide population are at risk for zinc deficiency and that zinc deficiency is associated with a variety of brain disorders and disease states in humans, it is of major interest to investigate, how these behavioral changes will affect the individual and a putative course of a disease. Thus, here, we provide a state of the art overview about the behavioral phenotypes observed in various models of zinc deficiency, among them environmentally produced zinc deficient animals as well as animal models based on a genetic alteration of a particular zinc homeostasis gene. Finally, we compare the behavioral phenotypes to the human condition of mild to severe zinc deficiency and provide a model, how zinc deficiency that is associated with many neurodegenerative and neuropsychological disorders might modify the disease pathologies.

A growing amount of research indicates that abnormalities in the gastrointestinal (GI) system during development might be a common factor in multiple neurological disorders and might be responsible for some of the shared comorbidities seen among these diseases. For example, many patients with Autism Spectrum Disorder (ASD) have symptoms associated with GI disorders. Maternal zinc status may be an important factor given the multifaceted effect of zinc on gut development and morphology in the offspring. Zinc status influences and is influenced by multiple factors and an interdependence of prenatal and early life stress, immune system abnormalities, impaired GI functions, and zinc deficiency can be hypothesized. In line with this, systemic inflammatory events and prenatal stress have been reported to increase the risk for ASD. Thus, here, we will review the current literature on the role of zinc in gut formation, a possible link between gut and brain development in ASD and other neurological disorders with shared comorbidities, and tie in possible effects on the immune system. Based on these data, we present a novel model outlining how alterations in the maternal zinc status might pathologically impact the offspring leading to impairments in brain functions later in life.

Various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders (ASD). Substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as Fe, Cu, Pb, Hg, Se, and Zn may mediate synaptic dysfunction and impair synapse formation and maturation. Here, we performed in vitro studies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons. We analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers. Moreover, we evaluated whether a biometal profile characteristic for ASD patients influences synapse formation, maturation, and composition regarding NMDA receptor subunits and Shank proteins. Our results show that an ASD like biometal profile leads to a reduction of NMDAR (NR/Grin/GluN) subunit 1 and 2a, as well as Shank gene expression along with a reduction of synapse density. Additionally, synaptic protein levels of GluN2a and Shanks are reduced. Although Zn supplementation is able to rescue the aforementioned alterations, Zn deficiency is not solely responsible as causative factor. Thus, we conclude that balancing Zn levels in ASD might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis.