Running in the FAMILY: understanding and predicting the intergenerational transmission of mental illness.

Over 50% of children with a parent with severe mental illness will develop mental illness by early adulthood. However, intergenerational transmission of risk for mental illness in one's children is insufficiently considered in clinical practice, nor is it sufficiently utilised into diagnostics and care for children of ill parents. This leads to delays in diagnosing young offspring and missed opportunities for protective actions and resilience strengthening. Prior twin, family, and adoption studies suggest that the aetiology of mental illness is governed by a complex interplay of genetic and environmental factors, potentially mediated by changes in epigenetic programming and brain development. However, how these factors ultimately materialise into mental disorders remains unclear. Here, we present the FAMILY consortium, an interdisciplinary, multimodal (e.g., (epi)genetics, neuroimaging, environment, behaviour), multilevel (e.g., individual-level, family-level), and multisite study funded by a European Union Horizon-Staying-Healthy-2021 grant. FAMILY focuses on understanding and prediction of intergenerational transmission of mental illness, using genetically informed causal inference, multimodal normative prediction, and animal modelling. Moreover, FAMILY applies methods from social sciences to map social and ethical consequences of risk prediction to prepare clinical practice for future implementation. FAMILY aims to deliver: (i) new discoveries clarifying the aetiology of mental illness and the process of resilience, thereby providing new targets for prevention and intervention studies; (ii) a risk prediction model within a normative modelling framework to predict who is at risk for developing mental illness; and (iii) insight into social and ethical issues related to risk prediction to inform clinical guidelines.

Role of CAMK2D in neurodevelopment and associated conditions.

The calcium/calmodulin-dependent protein kinase type 2 (CAMK2) family consists of four different isozymes, encoded by four different genes-CAMK2A, CAMK2B, CAMK2G, and CAMK2D-of which the first three have been associated recently with neurodevelopmental disorders. CAMK2D is one of the major CAMK2 proteins expressed in the heart and has been associated with cardiac anomalies. Although this CAMK2 isoform is also known to be one of the major CAMK2 subtypes expressed during early brain development, it has never been linked with neurodevelopmental disorders until now. Here we show that CAMK2D plays an important role in neurodevelopment not only in mice but also in humans. We identified eight individuals harboring heterozygous variants in CAMK2D who display symptoms of intellectual disability, delayed speech, behavioral problems, and dilated cardiomyopathy. The majority of the variants tested lead to a gain of function (GoF), which appears to cause both neurological problems and dilated cardiomyopathy. In contrast, loss-of-function (LoF) variants appear to induce only neurological symptoms. Together, we describe a cohort of individuals with neurodevelopmental disorders and cardiac anomalies, harboring pathogenic variants in CAMK2D, confirming an important role for the CAMK2D isozyme in both heart and brain function.

PSMC3 proteasome subunit variants are associated with neurodevelopmental delay and type I interferon production.

A critical step in preserving protein homeostasis is the recognition, binding, unfolding, and translocation of protein substrates by six AAA-ATPase proteasome subunits (ATPase-associated with various cellular activities) termed PSMC1-6, which are required for degradation of proteins by 26S proteasomes. Here, we identified 15 de novo missense variants in the PSMC3 gene encoding the AAA-ATPase proteasome subunit PSMC3/Rpt5 in 23 unrelated heterozygous patients with an autosomal dominant form of neurodevelopmental delay and intellectual disability. Expression of PSMC3 variants in mouse neuronal cultures led to altered dendrite development, and deletion of the PSMC3 fly ortholog Rpt5 impaired reversal learning capabilities in fruit flies. Structural modeling as well as proteomic and transcriptomic analyses of T cells derived from patients with PSMC3 variants implicated the PSMC3 variants in proteasome dysfunction through disruption of substrate translocation, induction of proteotoxic stress, and alterations in proteins controlling developmental and innate immune programs. The proteostatic perturbations in T cells from patients with PSMC3 variants correlated with a dysregulation in type I interferon (IFN) signaling in these T cells, which could be blocked by inhibition of the intracellular stress sensor protein kinase R (PKR). These results suggest that proteotoxic stress activated PKR in patient-derived T cells, resulting in a type I IFN response. The potential relationship among proteosome dysfunction, type I IFN production, and neurodevelopment suggests new directions in our understanding of pathogenesis in some neurodevelopmental disorders.

Adult Camk2a gene reinstatement restores the learning and plasticity deficits of Camk2a knockout mice.

With the recent findings that mutations in the gene encoding the α-subunit of calcium/calmodulin-dependent protein kinase II (CAMK2A) causes a neurodevelopmental disorder (NDD), it is of great therapeutic relevance to know if there exists a critical developmental time window in which CAMK2A needs to be expressed for normal brain development, or whether expression of the protein at later stages is still beneficial to restore normal functioning. To answer this question, we generated an inducible Camk2a mouse model, which allows us to express CAMK2A at any desired time. Here, we show that adult expression of CAMK2A rescues the behavioral and electrophysiological phenotypes seen in the Camk2a knock-out mice, including spatial and conditional learning and synaptic plasticity. These results suggest that CAMK2A does not play a critical irreversible role in neurodevelopment, which is of importance for future therapies to treat CAMK2A-dependent disorders.

Development of a medium throughput whole-cell microtiter plate Thr286 autophosphorylation assay for CaMKIIα using ELISA.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a multifunctional Ser/Thr kinase involved in several neuronal signaling pathways including synaptic plasticity. CaMKIIα autonomous activity is highly dependent on Thr286 autophosphorylation (pThr286), which is widely used as a readout for its enzymatic activity. To readily characterise compounds and potential drug candidates targeting CaMKIIα, a simple, generic cell-based assay for quantification of pThr286 levels is needed. In this study, we present a cell-based assay using an adapted ELISA as a suitable and higher throughput alternative to Western blotting. In this 96-well plate-based assay, we use whole HEK293T cells recombinantly expressing CaMKIIα and apply a phospho-specific antibody to detect pThr286 levels by chemiluminescence. In parallel, total CaMKIIα expression levels are detected by fluorescence using an Alexa488-conjugated anti-myc antibody targeting a C-terminal myc-tag. By multiplexing chemiluminescence and fluorescence, phosphorylation levels are normalised to CaMKIIα total expression within each well. The specificity of the assay was confirmed using a phosphodead mutant (T286A) of CaMKIIα. By applying Ca2+ or known CaMKIIα inhibitors (KN93, tatCN21 and AS100105) and obtaining concentration-response curves, we demonstrate high sensitivity and validity of the assay. Lastly, we demonstrate the versatility of the assay by determining autophosphorylation levels in CaMKIIα patient-related mutations, known to possess altered pThr286 responses (E109D, E183V and H282R). The established assay for CaMKIIα is a reproducible, easily implemented, and facile ELISA-based assay that allows for reliable quantification of pThr286 levels.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Case Report: Developmental Delay and Acute Neuropsychiatric Episodes Associated With a de novo Mutation in the CAMK2B Gene (c.328G>A p.Glu110Lys).

Mutations in the genes encoding calcium/calmodulin dependent protein kinase II (CAMK2) isoforms cause a newly recognized neurodevelopmental disorder (ND), for which the full clinical spectrum has yet to be described. Here we report the detailed description of a child with a de novo gain of function (GoF) mutation in the gene Ca/Calmodulin dependent protein kinase 2 beta (CAMK2B c.328G > A p.Glu110Lys) who presents with developmental delay and periodic neuropsychiatric episodes. The episodes manifest as encephalopathy with behavioral changes, headache, loss of language and loss of complex motor coordination. Additionally, we provide an overview of the effect of different medications used to try to alleviate the symptoms. We show that medications effective for mitigating the child's neuropsychiatric symptoms may have done so by decreasing CAMK2 activity and associated calcium signaling; whereas medications that appeared to worsen the symptoms may have done so by increasing CAMK2 activity and associated calcium signaling. We hypothesize that by classifying CAMK2 mutations as "gain of function" or "loss of function" based on CAMK2 catalytic activity, we may be able to guide personalized empiric treatment regimens tailored to specific CAMK2 mutations. In the absence of sufficient patients for traditional randomized controlled trials to establish therapeutic efficacy, this approach may provide a rational approach to empiric therapy for physicians treating patients with dysregulated CAMK2 and associated calcium signaling.

Focused ultrasound neuromodulation on a multiwell MEA.

BACKGROUND: Microelectrode arrays (MEA) enable the measurement and stimulation of the electrical activity of cultured cells. The integration of other neuromodulation methods will significantly enhance the application range of MEAs to study their effects on neurons. A neuromodulation method that is recently gaining more attention is focused ultrasound neuromodulation (FUS), which has the potential to treat neurological disorders reversibly and precisely. METHODS: In this work, we present the integration of a focused ultrasound delivery system with a multiwell MEA plate. RESULTS: The ultrasound delivery system was characterised by ultrasound pressure measurements, and the integration with the MEA plate was modelled with finite-element simulations of acoustic field parameters. The results of the simulations were validated with experimental visualisation of the ultrasound field with Schlieren imaging. In addition, the system was tested on a murine primary hippocampal neuron culture, showing that ultrasound can influence the activity of the neurons. CONCLUSIONS: Our system was demonstrated to be suitable for studying the effect of focused ultrasound on neuronal cultures. The system allows reproducible experiments across the wells due to its robustness and simplicity of operation.

Loss of CAMK2G affects intrinsic and motor behavior but has minimal impact on cognitive behavior.

Introduction: The gamma subunit of calcium/calmodulin-dependent protein kinase 2 (CAMK2G) is expressed throughout the brain and is associated with neurodevelopmental disorders. Research on the role of CAMK2G is limited and attributes different functions to specific cell types. Methods: To further expand on the role of CAMK2G in brain functioning, we performed extensive phenotypic characterization of a Camk2g knockout mouse. Results: We found different CAMK2G isoforms that show a distinct spatial expression pattern in the brain. Additionally, based on our behavioral characterization, we conclude that CAMK2G plays a minor role in hippocampus-dependent learning and synaptic plasticity. Rather, we show that CAMK2G is required for motor function and that the loss of CAMK2G results in impaired nest-building and marble burying behavior, which are innate behaviors that are associated with impaired neurodevelopment. Discussion: Taken together, our results provide evidence for a unique function of this specific CAMK2 isozyme in the brain and further support the role of CAMK2G in neurodevelopment.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

The Diagnostic Journey of a Patient with Prader-Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature.

Prader-Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader-Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the 'Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations' (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype-phenotype relationship in PWS.

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity.

Hyperactivation of the mammalian target of rapamycin (mTOR) pathway can cause malformation of cortical development (MCD) with associated epilepsy and intellectual disability (ID) through a yet unknown mechanism. Here, we made use of the recently identified dominant-active mutation in Ras Homolog Enriched in Brain 1 (RHEB), RHEBp.P37L, to gain insight in the mechanism underlying the epilepsy caused by hyperactivation of the mTOR pathway. Focal expression of RHEBp.P37L in mouse somatosensory cortex (SScx) results in an MCD-like phenotype, with increased mTOR signaling, ectopic localization of neurons, and reliable generalized seizures. We show that in this model, the mTOR-dependent seizures are caused by enhanced axonal connectivity, causing hyperexcitability of distally connected neurons. Indeed, blocking axonal vesicle release from the RHEBp.P37L neurons alone completely stopped the seizures and normalized the hyperexcitability of the distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, which can lead to generalized epilepsy.

Role of calcium/calmodulin-dependent kinase 2 in neurodevelopmental disorders.

Neurodevelopmental disorders are a complex and heterogeneous group of neurological disorders characterized by their early-onset and estimated to affect more than 3% of children worldwide. The rapid advancement of sequencing technologies in the past years allowed the identification of hundreds of variants in several different genes causing neurodevelopmental disorders. Between those, new variants in the Calcium/calmodulin dependent protein kinase II (CAMK2) genes were recently linked to intellectual disability. Despite many years of research on CAMK2, this proves for the first time that this well-known and highly conserved molecule plays an important role in the human brain. In this review, we give an overview of the identified CAMK2 variants, and we speculate on potential mechanisms through which dysfunctions in CAMK2 result in neurodevelopmental disorders. Additionally, we discuss how the identification of CAMK2 variants might result in new exciting discoveries regarding the function of CAMK2 in the human brain.

TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.

Assessing the requirements of prenatal UBE3A expression for rescue of behavioral phenotypes in a mouse model for Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of functional ubiquitin protein ligase E3A (UBE3A). In neurons, UBE3A expression is tightly regulated by a mechanism of imprinting which suppresses the expression of the paternal UBE3A allele. Promising treatment strategies for AS are directed at activating paternal UBE3A gene expression. However, for such strategies to be successful, it is important to know when such a treatment should start, and how much UBE3A expression is needed for normal embryonic brain development. METHODS: Using a conditional mouse model of AS, we further delineated the critical period for UBE3A expression during early brain development. Ube3a gene expression was induced around the second week of gestation and mouse phenotypes were assessed using a behavioral test battery. To investigate the requirements of embryonic UBE3A expression, we made use of mice in which the paternal Ube3a allele was deleted. RESULTS: We observed a full behavioral rescue of the AS mouse model phenotypes when Ube3a gene reactivation was induced around the start of the last week of mouse embryonic development. We found that full silencing of the paternal Ube3a allele was not completed till the first week after birth but that deletion of the paternal Ube3a allele had no significant effect on the assessed phenotypes. LIMITATIONS: Direct translation to human is limited, as we do not precisely know how human and mouse brain development aligns over gestational time. Moreover, many of the assessed phenotypes have limited translational value, as the underlying brain regions involved in these tasks are largely unknown. CONCLUSIONS: Our findings provide further important insights in the requirement of UBE3A expression during brain development. We found that loss of up to 50% of UBE3A protein during prenatal mouse brain development does not significantly impact the assessed mouse behavioral phenotypes. Together with previous findings, our results indicate that the most critical function for mouse UBE3A lies in the early postnatal period between birth and P21.

CaMKII controls neuromodulation via neuropeptide gene expression and axonal targeting of neuropeptide vesicles.

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.

Netrin-G2 dysfunction causes a Rett-like phenotype with areflexia.

We describe the underlying genetic cause of a novel Rett-like phenotype accompanied by areflexia in three methyl-CpG-binding protein 2-negative individuals from two unrelated families. Discovery analysis was performed using whole-exome sequencing followed by Sanger sequencing for validation and segregation. Functional studies using short-hairpin RNA for targeted gene knockdown were implemented by the transfection of mouse cultured primary hippocampal neurons and in vivo by in utero electroporation. All patients shared a common homozygous frameshift mutation (chr9:135073515, c.376dupT, p.(Ser126PhefsTer241)) in netrin-G2 (NTNG2, NM_032536.3) with predicted nonsense-mediated decay. The mutation fully segregated with the disease in both families. The knockdown of either NTNG2 or the related netrin-G family member NTNG1 resulted in severe neurodevelopmental defects of neuronal morphology and migration. While NTNG1 has previously been linked to a Rett syndrome (RTT)-like phenotype, this is the first description of a RTT-like phenotype caused by NTNG2 mutation. Netrin-G proteins have been shown to be required for proper axonal guidance during early brain development and involved in N-methyl- d-aspartate-mediated synaptic transmission. Our results demonstrating that knockdown of murine NTNG2 causes severe impairments of neuronal morphology and cortical migration are consistent with those of RTT animal models and the shared neurodevelopmental phenotypes between the individuals described here and typical RTT patients.

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons.

Background: Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods: Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results: We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber-binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions: We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

The Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes.

Tunneling nanotubes (TNTs) are actin-based transient tubular connections that allow direct communication between distant cells. TNTs play an important role in several physiological (development, immunity, and tissue regeneration) and pathological (cancer, neurodegeneration, and pathogens transmission) processes. Here, we report that the Wnt/Ca2+ pathway, an intracellular cascade that is involved in actin cytoskeleton remodeling, has a role in TNT formation and TNT-mediated transfer of cargoes. Specifically, we found that Ca2+ /calmodulin-dependent protein kinase II (CaMKII), a transducer of the Wnt/Ca2+ pathway, regulates TNTs in a neuronal cell line and in primary neurons. We identified the ß isoform of CaMKII as a key molecule in modulating TNT formation and transfer, showing that this depends on the actin-binding activity of the protein. Finally, we found that the transfer of vesicles and aggregated α-synuclein between primary neurons can be regulated by the activation of the Wnt/Ca2+ pathway. Our findings suggest that Wnt/Ca2+ pathway could be a novel promising target for therapies designed to impair TNT-mediated propagation of pathogens.

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-ß (CaMKII-ß) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-ß Antibody With Connexin36.

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-ß at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-ß. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-ß-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-ß. However, a monoclonal antibody (CB-ß-1) recognized CaMKII-ß but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-ß expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-ß also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-ß influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-ß-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-ß is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-ß with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-ß may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.