Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications.

Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as "genetic scissors". One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery.

Enigmatic rhodopsin mutation creates an exceptionally strong splice acceptor site.

The c.620 T > G mutation in rhodopsin found in the first mapped autosomal dominant retinitis pigmentosa (adRP) locus is associated with severe, early-onset RP. Intriguingly, another mutation affecting the same nucleotide (c.620 T > A) is related to a mild, late-onset RP. Assuming that both mutations are missense mutations (Met207Arg and Met207Lys) hampering the ligand-binding pocket, previous work addressed how they might differentially impair rhodopsin function. Here, we investigated the impact of both mutations at the mRNA and protein level in HEK293 cells and in the mouse retina. We show that, in contrast to c.620 T > A, c.620 T > G is a splicing mutation, which generates an exceptionally strong splice acceptor site (SAS) resulting in a 90 bp in-frame deletion and protein mislocalization in vitro and in vivo. Moreover, we identified the core element underlying the c.620 T > G SAS strength. Finally, we demonstrate that the c.620 T > G SAS is very flexible in branch point choice, which might explain its remarkable performance. Based on these results, we suggest that (i) point mutations should be routinely tested for mRNA splicing to avoid dispensable analysis of mutations on protein level, which do not naturally exist. (ii) Puzzling disease courses of mutations in other genes might also correlate with their effects on mRNA splicing. (iii) Flexibility in branch point choice might be another factor influencing the SAS strength. (iv) The core splice element identified in this study could be useful for biotechnological applications requiring effective SAS.

Preliminary Assessment of Pre-Electroconvulsive Therapy Evaluation Practices in European Countries: The Need for Guidelines.

OBJECTIVES: Pre-electroconvulsive therapy (ECT) evaluation is an essential part of ECT preparation, a standard treatment in the psychiatric field. However, no routine pre-ECT evaluation has been published so far. This preliminary study aimed to explore different practices in pre-ECT evaluation across European countries. METHODS: The data were collected as a snowball sample approach using an online survey from September 2019 to April 2020. The final analysis included data from 18 clinics placed in 16 European countries. RESULTS: Regulations on the pre-ECT evaluation were found in 9 countries. All clinics reported doing complete blood count, serum electrolytes, and renal function analysis as a part of regular laboratory testing, alongside with a cardiovascular assessment. Ten clinics reported using psychiatric scales. Six clinics reported doing a cognitive assessment, of which all had regulations on the pre-ECT evaluation. Not one evaluation had the same sets of procedures and diagnostics. CONCLUSIONS: The differences in assessment approaches mirror high variability of the pre-ECT evaluation practice across Europe. Cognitive assessment and objectification of psychiatric symptoms should be a regular part of the pre-ECT evaluation because of the monitoring of the most common adverse effect and observing the clinical response to ECT. Standardization of the pre-ECT evaluation and ECT in general would remove criticisms and opposition to the treatment, make it based on the best of our knowledge, and provide a method respectful of patients' best interests and rights.

Preformative, Performative and Narative in Creative Person Centered Psychopharmacotherapy.

Psychopharmacotherapy does not stand alone. The act of prescribing involves much more than solely choosing "best" medication. It seems that somewhere in the process of trying to objectify and scientify our therapy, we have neglected an important and effective dimensions of it. Psychopharmacology should consider much more than just biological dimension of drugs. Psychological, social and behavioral factors that influence drug metabolism, efficacy and side-effects are largely overlooked. Obviously, the subtext of information provided by the medical professional inevitably contains suggestion. Important part of that subtext is consisted in way we think of it, we talk of it and we perform that information. Defining of preformative and performative psychopharmacotherapy was atempted as well as desciption of narrative creative person-centered psychopharmacotherapy. Studies that indicate that medicines (SSRI) do not work on its own but as amplifier of the influence of the living conditions on mood are provided. Undirected susceptibility to change hypothesis which request acknowledging the importance of social, psychological, environmental factors to explain such the mechanisms underlying the recovery from the disease is explained. Understanding the role of medicines (SSRIs) as amplifier of the influence of the living conditions on mood represents a critical step in developing a creative, person-centered psychopharmacotherapy aimed at better matching patients with treatment and avoiding potential harmful consequences.

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

Bihavioral Addictions in Childhood and Adolescence - Pandemic Knocking Door.

INTRODUCTION: Addiction is not solely "substance dependence". Diminished control is a core defining concept of psychoactive substance addiction. Several behaviors, besides psychoactive substance ingestion, produce short-term reward that may engender diminished control over the behavior. Growing evidence suggests that behavioral addictions resemble substance addictions in many domains, including phenomenology, tolerance, comorbidity, overlapping genetic contribution, neurobiological mechanisms, and response to treatment. This similarity has given rise to the concept of non-substance or behavioral addictions, i.e., syndromes analogous to substance addiction, but with a behavioral focus. The type of excessive behaviors identified as being addictive include gambling, use of computers, playing video games, use of the internet, exercise, and shopping. Behavioral addictions have been proposed as a new class in DSM-5, but the only category included is gambling disorder. Internet gaming disorder is included in the appendix as a condition for further study. The ICD-11 included also the definition of a new disorder, gaming disorder. To present actual knowledge about behavioral addictions in childhood and adolescence. METHODS: Analysis of data in available literature in data basis and textbooks. RESULTS: Some behavioral addictions are becoming more common in children and adolescents. Dominant are gaming and gambling addiction that are also best researched. CONCLUSIONS: Behavioral addiction becomes an epidemic in children and adolescents.

Psychological Disorders in Childhood and Adolescent Age - New Classifications.

INTRODUCTION: The eleventh revision of the International Classification of Diseases (ICD-11) is planned to be published in 2018. So called, "beta version" of the chapter of mental and behavioral disorders (ICD-11) is already available and it is considered that there will be no significant deviations in the final version. The DSM-5 was released in 2013. Changes related to mental disorders in child and adolescent psychiatry have been made in both of these classifications. To identify changes in the classifications of mental disorders in childhood and adolescent age in beta version of ICD-11 and DSM-5. METHODS: Review of mental disorders in childhood and adolescent age and their classification in ICD-11 and DSM-5. RESULTS: For disorders that are classified as "mental retardation" in ICD-10, a new term "intellectual development disorders" has been introduced in ICD-11, ie "intellectual disabilities" in DSM-5. Hyperactivity disorders and attention deficit is a separate entity in relation to ICD-10, in which it is classified as a hyperkinetic disorder. Asperger's syndrome, which is isolated from autism spectrum disorders in DSM-5, does not appear under that name in ICD-11 either. Elimination disorders are in a separate block MKB-11 and DSM-5. Speech and language disorders are classified as communication disorders in the DSM-5 classification. Selective mutism and anxiety separation disorder in childhood are in the block of anxiety and fear-related disorders in ICD-11, and among anxiety disorders in DSM-5, respectively. Reactive emotional disorder and disinhibited attachment disorder of childhood are classified as stress-related disorders in ICD-11 and DSM-5. CONCLUSIONS: The new classifications (ICD-11 and DSM-5) classify mental disorders in child and adolescent psychiatry somewhat differently from their antecedents. New entities have also been formed.

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.

Sexual Dysfunctions in Bosnian War Veterans.

Sexual functioning of war veterans is significantly under-explored. During devastating aggression on Bosnia-Herzegovina (BiH) around 400 thousand soldiers were included in combats. It is estimated that more than 100 000 persons were killed, and more than 60 000 them were soldiers. Vast majority of them were deployed since war is ended. We found high prevalence of sexual dysfunctions in war veterans. Also significant difference in several areas of sexual functioning between war veterans with and without symptoms of posttraumatic stress disorder was found.

Peripherin-2 differentially interacts with cone opsins in outer segments of cone photoreceptors.

Peripherin-2 is a glycomembrane protein exclusively expressed in the light-sensing compartments of rod and cone photoreceptors designated as outer segments (OS). Mutations in peripherin-2 are associated with degenerative retinal diseases either affecting rod or cone photoreceptors. While peripherin-2 has been extensively studied in rods, there is only little information on its supramolecular organization and function in cones. Recently, we have demonstrated that peripherin-2 interacts with the light detector rhodopsin in OS of rods. It remains unclear, however, if peripherin-2 also binds to cone opsins. Here, using a combination of co-immunoprecipitation analyses, transmission electron microscopy (TEM)-based immunolabeling experiments, and quantitative fluorescence resonance energy transfer (FRET) measurements in cone OS of wild type mice, we demonstrate that peripherin-2 binds to both, S-opsin and M-opsin. However, FRET-based quantification of the respective interactions indicated significantly less stringent binding of peripherin-2 to S-opsin compared to its interaction with M-opsin. Subsequent TEM-studies also showed less co-localization of peripherin-2 and S-opsin in cone OS compared to peripherin-2 and M-opsin. Furthermore, quantitative FRET analysis in acutely isolated cone OS revealed that the cone degeneration-causing V268I mutation in peripherin-2 selectively reduced binding to M-opsin without affecting the peripherin-2 interaction to S-opsin or rhodopsin. The differential binding of peripherin-2 to cone opsins and the mutant-specific interference with the peripherin-2/M-opsin binding points to a novel role of peripherin-2 in cones and might contribute to understanding the differential penetrance of certain peripherin-2 mutations in rods and cones. Finally, our results provide a proof-of-principle for quantitative FRET measurements of protein-protein interactions in cone OS.

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

Gene therapy for achromatopsia.

The present review summarizes the current status of achromatopsia (ACHM) gene therapy-related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide-gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno-associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy.

Family Atmosphere and Relationships as Predictors of Heroin Addiction.

INTRODUCTION: Studies show that dysfunctional family relationships are important predictors of addictions to all psychoactive substances. OBJECTIVE: To establish if there is a connection between family relations and heroin addiction and if found to exist, what is the quality of this connection. SUBJECTS AND METHODS: This research was conducted on the sample comprised of 160 subjects divided into two groups. The first group consisted of 61 heroin addicts treated at the Tuzla University Clinical Centre Psychiatric Hospital. The second group consisted of 99 subjects who were students at the Tuzla University Faculties of Philosophy and Electrical Engineering and who were not using any psychoactive substances. The subjects were tested with the Quality of Family Interactions Scale (KOBI) which measures the interactions between children and parents in two dimensions, described in literature as 'acceptance' and 'rejection'. RESULTS: The research team established statistically significant differences between the heroin addicts and the students, the non-users, in terms of their family relationships. The results show that the addicts families were characterized by lack of understanding, by conflicts, rejection, non-acceptance by parents, while the non-users families were characterized by understanding, acceptance by parents and good communication. CONCLUSIONS: There is a connection between inter-family relationships and addiction. Namely, rejection and non-acceptance of children/persons by their families and parents, bad communication and dysfunctional family relationships are significant predictors of heroin addiction.

Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors.

Outer segments (OSs) of rod photoreceptors are cellular compartments specialized in the conversion of light into electrical signals. This process relies on the light-triggered change in the intracellular levels of cyclic guanosine monophosphate, which in turn controls the activity of cyclic nucleotide-gated (CNG) channels in the rod OS plasma membrane. The rod CNG channel is a macromolecular complex that in its core harbors the ion-conducting CNGA1 and CNGB1a subunits. To identify additional proteins of the complex that interact with the CNGB1a core subunit, we applied affinity purification of mouse retinal proteins followed by mass spectrometry. In combination with in vitro and in vivo co-immunoprecipitation and fluorescence resonance energy transfer (FRET), we found that the tetraspanin peripherin-2 links CNGB1a to the light-detector rhodopsin. Using immunoelectron microscopy, we found that this peripherin-2/rhodopsin/CNG channel complex localizes to the contact region between the disk rims and the plasma membrane. FRET measurements revealed that the fourth transmembrane domain (TM4) of peripherin-2 is required for the interaction with rhodopsin. Quantitatively, the binding affinity of the peripherin-2/rhodopsin interaction was in a similar range as that observed for rhodopsin dimers. Finally, we demonstrate that the p.G266D retinitis pigmentosa mutation found within TM4 selectively abolishes the binding of peripherin-2 to rhodopsin. This finding suggests that the specific disruption of the rhodopsin/peripherin-2 interaction in the p.G266D mutant might contribute to the pathophysiology in affected persons.

Metabolic syndrome in schizophrenia - who is more to blame: FGA polypharmacy or clozapine monotherapy?

BACKGROUND: To establish the prevalence of metabolic syndrome and its parameters in group of patients with schizophrenia in polypharmacy - receiving first generation antipsychotics versus clozapine alone treated group. SUBJECTS AND METHODS: 48 outpatients with schizophrenia divided into two groups: the first group of 21 patients in polypharmacy with first generation antipsychotics, and the second group of 27 patients treated with clozapine alone were assessed for the presence of metabolic syndrome. We used logistic regression models to assess the relationship between metabolic syndrome and antipsychotic therapy, gender and age. RESULTS: Metabolic syndrome was found in 52.1% of all subjects. Compared to first generation antipsychotics polypharmacy, the monopharmacy with clozapine was associated with elevated rates of metabolic syndrome (28.6% vs. 70.4%, p=0.004). With regard to particular parameters of metabolic syndrome, the elevated plasma triglycerides were significantly more present in subjects within Clozapine group (p=0.03). Logistic regression analysis showed that female gender (p=0.004), and clozapine treatment (p=0.005) were significantly associated with metabolic syndrome. CONCLUSION: Compared to polypharmacy with first generation antipsychotics, the higher prevalence of metabolic syndrome is found in patients treated with Clozapine alone. The most prevalent metabolic disorder is dyslipidemia.

Sick sinus syndrome in HCN1-deficient mice.

BACKGROUND: Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND. METHODS AND RESULTS: Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output. CONCLUSIONS: We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.