Generation of a human induced pluripotent stem cell line JHUi004-A with heterozygous mutation for spinocerebellar ataxia type 12 using genome editing.

Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9n genome editing, we have generated a human heterozygous SCA12 iPSC line with 73 triplets for the mutant allele. The heterozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Role of Bß1 overexpression in the pathogenesis of SCA12.

BACKGROUND: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by a CAG/CTG repeat expansion at the PPP2R2B locus. OBJECTIVE: We investigated how the CAG repeat expansion within the PPP2R2B 7B7D transcript influences the expression of Bß1 and a potential protein containing a long polyserine tract. METHODS: Transcript and protein expression were measured using quantitative PCR (qPCR) Role of Bß1 overexpression in the pathogenesis of SCA12 and Western blot, respectively, in an SK-N-MC cell model that overexpresses the full-length PPP2R2B 7B7D transcript. The apoptotic effect of a protein containing a long polyserine tract on SK-N-MC cells was evaluated using caspase 3/7 activity. RESULTS: The CAG repeat expansion increases the expression of the PPP2R2B 7B7D transcript, as well as Bß1 protein, in an SK-N-MC cell model in which the full-length PPP2R2B 7B7D transcript is overexpressed. The CAG repeat expansion within the 7B7D transcript is translated into a long polyserine tract that triggers apoptosis in SK-N-MC cells. CONCLUSIONS: The SCA12 mutation leads to overexpression of PPP2R2B Bß1 and to expression of a protein containing a long polyserine tract; both these effects potentially contribute to SCA12 pathogenesis. © 2024 International Parkinson and Movement Disorder Society.

Tobacco smoking and nicotine vaping in persons with first episode psychosis.

Tobacco smoking is highly prevalent in persons with psychosis and is the leading cause of preventable mortality in this population. Less is known about tobacco smoking in persons with first episode psychosis (FEP) and there have been no estimates about the prevalence of nicotine vaping in FEP. This study reports rates of tobacco smoking and nicotine vaping in young people with FEP enrolled in Coordinated Specialty Care programs in Pennsylvania and Maryland. Using data collected from 2021 to 2023, we examined lifetime and recent smoking and vaping and compared smokers and vapers to nonusers on symptoms, functioning, and substance use. The sample included 445 participants aged 13-35 with recent psychosis onset. Assessments were collected by program staff. Overall, 28 % of participants engaged in either smoking or vaping within 30 days of the admission assessment. Smokers and vapers were disproportionately male, cannabis users, and had lower negative symptom severity than non-smokers. Vapers had higher role and social functioning. Both smoking and vaping were related to a longer time from psychosis onset to program enrollment. We compare these findings to previous studies and suggest steps for addressing smoking and vaping in this vulnerable population.

Huntington disease-like 2: insight into neurodegeneration from an African disease.

Huntington disease (HD)-like 2 (HDL2) is a rare genetic disease caused by an expanded trinucleotide repeat in the JPH3 gene (encoding junctophilin 3) that shows remarkable clinical similarity to HD. To date, HDL2 has been reported only in patients with definite or probable African ancestry. A single haplotype background is shared by patients with HDL2 from different populations, supporting a common African origin for the expansion mutation. Nevertheless, outside South Africa, reports of patients with HDL2 in Africa are scarce, probably owing to limited clinical services across the continent. Systematic comparisons of HDL2 and HD have revealed closely overlapping motor, cognitive and psychiatric features and similar patterns of cerebral and striatal atrophy. The pathogenesis of HDL2 remains unclear but it is proposed to occur through several mechanisms, including loss of protein function and RNA and/or protein toxicity. This Review summarizes our current knowledge of this African-specific HD phenocopy and highlights key areas of overlap between HDL2 and HD. Given the aforementioned similarities in clinical phenotype and pathology, an improved understanding of HDL2 could provide novel insights into HD and other neurodegenerative and/or trinucleotide repeat expansion disorders.

Bidirectional Transcription at the PPP2R2B Gene Locus in Spinocerebellar Ataxia Type 12.

BACKGROUND: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in the PPP2R2B gene. OBJECTIVE: In this study, we tested the hypothesis that the PPP2R2B antisense (PPP2R2B-AS1) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis. METHODS: Expression of PPP2R2B-AS1 transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific reverse transcription polymerase chain reaction. The tendency of expanded PPP2R2B-AS1 (expPPP2R2B-AS1) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescence in situ hybridization. The apoptotic effect of expPPP2R2B-AS1 transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated translation of expPPP2R2B-AS1 transcript in SK-N-MC cells. RESULTS: The repeat region in the PPP2R2B gene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. Transfected expPPP2R2B-AS1 transcripts induce apoptosis in SK-N-MC cells, and the apoptotic effect may be mediated, at least in part, by the RNA secondary structure. The expPPP2R2B-AS1 transcripts form CUG RNA foci in SK-N-MC cells. expPPP2R2B-AS1 transcript is translated in the alanine open reading frame (ORF) via repeat-associated non-ATG translation, which is diminished by single-nucleotide interruptions within the CUG repeat and MBNL1 overexpression. CONCLUSIONS: These findings suggest that PPP2R2B-AS1 contributes to SCA12 pathogenesis and may therefore provide a novel therapeutic target for the disease. © 2023 International Parkinson and Movement Disorder Society.

Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12.

OBJECTIVE: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in the PPP2R2B gene . Here we tested the hypothesis that the PPP2R2B antisense ( PPP2R2B-AS1 ) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis. METHODS: Expression of PPP2R2B-AS1 transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific RT-PCR (SS-RT-PCR). The tendency of expanded PPP2R2B-AS1 ( expPPP2R2B-AS1 ) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescence in situ hybridization. The toxic effect of expPPP2R2B-AS1 transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated (RAN) translation of expPPP2R2B-AS1 transcript in SK-N-MC cells. RESULTS: The repeat region in PPP2R2B gene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. Transfected expPPP2R2B-AS1 transcripts are toxic to SK-N-MC cells, and the toxicity may be mediated, at least in part, by the RNA secondary structure. The expPPP2R2B-AS1 transcripts form CUG RNA foci in SK-N-MC cells. expPPP2R2B-AS1 transcript is translated in the Alanine ORF via repeat-associated non-ATG (RAN) translation, which is diminished by single nucleotide interruptions within the CUG repeat, and MBNL1 overexpression. INTERPRETATION: These findings suggest that PPP2R2B-AS1 contributes to SCA12 pathogenesis, and may therefore provide a novel therapeutic target for the disease.

Parental perceptions of second opinion consultations for recent onset schizophrenia.

AIM: Correct early diagnosis and intervention for schizophrenia is associated with improved outcomes. This study sought to determine the potential value of specialized consultations for individuals thought to have a recent-onset schizophrenia spectrum disorder who were not receiving specialized care. METHODS: 121 consecutive one-time consultations were performed in a clinic specializing in recent-onset schizophrenia. Parents of 79 patients were questioned about the process and the value of the consultation. RESULTS: Consultations were perceived by parents as both positive and helpful experiences. 85% of parents were somewhat or very happy with the consultation experience, and 86% found the consultation to be moderately or very helpful. A third of parents viewed the consultation as contributing to the long-term improvement of the patient's condition. CONCLUSIONS: The findings demonstrate the potential value of consultations for individuals with possible schizophrenia spectrum disorders not receiving specialized treatment, and provide rationale for comprehensive studies investigating the widespread implementation of such consultations.

The Protective Effect of Clozapine on Suicide: A Population Mortality Study of Statewide Autopsy Records in Maryland.

Objective: Clozapine is the most efficacious antipsychotic medication, but it is underutilized and its mechanism of action is still poorly understood. One aspect of its unique efficacy that requires further study is its effect on suicidality. A randomized controlled trial, the InterSePT study, yielded evidence that clozapine reduces suicidality more than olanzapine, after which it became the only medication indicated for recurrent suicidal behavior in schizophrenia and schizoaffective disorder. We present here the first study of population mortality data to investigate the effect of clozapine on suicide.Methods: We reviewed statewide autopsy records of Maryland's Office of the Chief Medical Examiner, which performs uniquely comprehensive death investigations that include full toxicologic panels with postmortem blood levels of antipsychotics. Our study compared clozapine- and olanzapine-positive decedents across demographic, clinical, and manner-of-death outcomes using contingency table analysis and logistic regression.Results: Of 53,144 decedents from 2003 to 2021, 621 had clozapine or olanzapine detected on autopsy, with the two groups showing no demographic differences. Decedents with clozapine were significantly less likely to have died by suicide than by accident compared to those with olanzapine (odds ratio = 0.47; 95% CI, 0.26-0.84; P = .011).Conclusions: Our study thus adds more naturalistic evidence to the growing literature on the beneficial effect of clozapine on suicidality. Our findings also highlight the utility of statewide autopsy records, an untapped resource for investigating the potential protective effect of psychiatric medications on suicide at a population level.

Mutation of GPR143 Associated With Ocular Albinism Type 1, Intellectual Disability, and Schizophrenia: The Complex Biological and Social Interactions Between Genetic Syndromes and Mental Illness.

Copy number variations, which manifest primarily as deletions and duplications, contribute significantly to the genetic risk of schizophrenia. Specific syndromes associated with copy number variations, exemplified by the 22q11 deletion syndrome, confer both congenital abnormalities and an elevated risk of schizophrenia. We report the case of a patient with a deletion of exons 2 through 8 of GPR143. In addition to having the ophthalmologic disorder ocular albinism type 1 (OA1), a well-established consequence of mutations of GPR143, the patient is also intellectually impaired and impulsive, and he developed schizophrenia at age 15. Psychiatric manifestations of OA1 have not previously been reported, yet remain plausible, as the GPR143 protein is widely distributed in the brain and may function as an L-DOPA receptor. However, the patient described here also had a family history of psychiatric disorders independent of OA1, in utero exposure to heroin and cocaine, and challenging family circumstances. We suggest that the relationship between his GPR143 mutation and his psychiatric disorders is complex. The mutation may have directly contributed to his cognitive and psychiatric disorders, but we also suspect that OA1, present in multiple family members, contributed to multigenerational familial instability. Further, OA1 likely exacerbated our patient's cognitive and social impairment by interfering with his education, while his neuropsychiatric status frequently interfered with the assessment and treatment of his OA1. We conclude that the psychiatric and nonpsychiatric manifestations of a genetic syndrome are best managed in parallel and that psychiatrists and other mental health providers may be in the best position to assure that patients receive appropriate genetic and medical care.

RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Generation of a human induced pluripotent stem cell line JHUi003-A with homozygous mutation for spinocerebellar ataxia type 12 using genome editing.

Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding a brain-specific regulatory unit of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9 genome editing, we have generated a human homozygous SCA12 iPSC line with 69 and 72 triplets for each allele. The homozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs.

Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable "on-target" indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without "on target" indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

Hearing Voices and Seeing Things: Symptoms of Anxiety Misconstrued as Evidence of Schizophrenia in an Adolescent.

A patient's complaint of "hearing voices" or "seeing things" or of similar perceptual abnormalities leaves the clinician with 2 decisions: (1) Is the patient actually experiencing a hallucination, or does the complaint reflect a different mental experience, ranging from outright fabrication to the misinterpretation or mislabeling of vivid thoughts and emotions? (2) How should the experience reported by the patient, whether determined to be a hallucination or not, be understood in the context of the patient's entire history and mental state? We report the case of a 16-year-old whose cartoon-like hallucinations had led to the diagnosis of schizophrenia and had directed attention of the patient, her parents, and her clinicians away from critical issues of anxiety, depression, learning difficulties, and traumatic school experiences. This case illustrates how the diagnosis of schizophrenia can be driven by the prominence and vividness of psychotic-like symptoms reported by a patient, the expectation that patients' chief complaints must be directly and immediately addressed, insufficient attention to collateral information, and the distortions of a "checklist" approach to psychiatric diagnosis driven by the criteria in the Diagnostic and Statistical Manual of Mental Disorders, insurers, and the properties of electronic medical records. Given the consequences of either underdiagnosing or overdiagnosing schizophrenia, and the current lack of validated objective tests to assist with this diagnosis, clinicians are obligated to perform a thorough clinical assessment of such patients, including a probing exploration of the patient's mental state and a systematic collection of collateral information.

Pharmacological rescue in patient iPSC and mouse models with a rare DISC1 mutation.

We previously identified a causal link between a rare patient mutation in DISC1 (disrupted-in-schizophrenia 1) and synaptic deficits in cortical neurons differentiated from isogenic patient-derived induced pluripotent stem cells (iPSCs). Here we find that transcripts related to phosphodiesterase 4 (PDE4) signaling are significantly elevated in human cortical neurons differentiated from iPSCs with the DISC1 mutation and that inhibition of PDE4 or activation of the cAMP signaling pathway functionally rescues synaptic deficits. We further generated a knock-in mouse line harboring the same patient mutation in the Disc1 gene. Heterozygous Disc1 mutant mice exhibit elevated levels of PDE4s and synaptic abnormalities in the brain, and social and cognitive behavioral deficits. Pharmacological inhibition of the PDE4 signaling pathway rescues these synaptic, social and cognitive behavioral abnormalities. Our study shows that patient-derived isogenic iPSC and humanized mouse disease models are integral and complementary for translational studies with a better understanding of underlying molecular mechanisms.

A high-throughput screening to identify small molecules that suppress huntingtin promoter activity or activate huntingtin-antisense promoter activity.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). While there are currently no disease-modifying treatments for HD, recent efforts have focused on the development of nucleotide-based therapeutics to lower HTT expression. As an alternative to siRNA or oligonucleotide methods, we hypothesized that suppression of HTT expression might be accomplished by small molecules that either (1) directly decrease HTT expression by suppressing HTT promoter activity or (2) indirectly decrease HTT expression by increasing the promoter activity of HTT-AS, the gene antisense to HTT that appears to inhibit expression of HTT. We developed and employed a high-throughput screen for modifiers of HTT and HTT-AS promoter activity using luminescent reporter HEK293 cells; of the 52,041 compounds tested, we identified 898 replicable hits. We used a rigorous stepwise approach to assess compound toxicity and the capacity of the compounds to specifically lower huntingtin protein in 5 different cell lines, including HEK293 cells, HD lymphoblastoid cells, mouse primary neurons, HD iPSCs differentiated into cortical-like neurons, and HD hESCs. We found no compounds which were able to lower huntingtin without lowering cell viability in all assays, though the potential efficacy of a few compounds at non-toxic doses could not be excluded. Our results suggest that more specific targets may facilitate a small molecule approach to HTT suppression.

Impaired response of cerebral oxygen metabolism to visual stimulation in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG triplet repeat expansion in the Huntingtin gene. Metabolic and microvascular abnormalities in the brain may contribute to early physiological changes that subserve the functional impairments in HD. This study is intended to investigate potential abnormality in dynamic changes in cerebral blood volume (CBV) and cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO2) in the brain in response to functional stimulation in premanifest and early manifest HD patients. A recently developed 3-D-TRiple-acquisition-after-Inversion-Preparation magnetic resonance imaging (MRI) approach was used to measure dynamic responses in CBV, CBF, and CMRO2 during visual stimulation in one single MRI scan. Experiments were conducted in 23 HD patients and 16 healthy controls. Decreased occipital cortex CMRO2 responses were observed in premanifest and early manifest HD patients compared to controls (P < 0.001), correlating with the CAG-Age Product scores in these patients (R2 = 0.4, P = 0.001). The results suggest the potential value of this reduced CMRO2 response during visual stimulation as a biomarker for HD and may illuminate the role of metabolic alterations in the pathophysiology of HD.

Research Domain Criteria: Strengths, Weaknesses, and Potential Alternatives for Future Psychiatric Research.

The Research Domain Criteria (RDoC) paradigm was launched 10 years ago as a superior approach for investigation of mental illness. RDoC conceptualizes normal human behavior, emotion, and cognition as dimensional, with mental illnesses as dimensional extremes. We suggest that RDoC may have value for understanding normal human psychology and some conditions plausibly construed as extremes of normal variation. By contrast, for the most serious of mental illnesses, including dementia, autism, schizophrenia, and bipolar disorder, we argue that RDoC is conceptually flawed. RDoC conflates variation along dimensional axes of normal function with quantitative measurements of disease phenotypes and with the occurrence of diseases in overlapping clusters or spectra. This moves away from the disease model of major mental illness. Further, RDoC imposes a top-down approach to research. We argue that progress in major mental illness research will be more rapid with a bottom-up approach, starting with the discovery of etiological factors, proceeding to investigation of pathogenic pathways, including use of cell and animal models, and leading to a refined nosology and novel, targeted treatments.

Differential Changes in Functional Connectivity of Striatum-Prefrontal and Striatum-Motor Circuits in Premanifest Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder. The striatum is one of the first brain regions that show detectable atrophy in HD. Previous studies using functional magnetic resonance imaging (fMRI) at 3 tesla (3 T) revealed reduced functional connectivity between striatum and motor cortex in the prodromal period of HD. Neuroanatomical and neurophysiological studies have suggested segregated corticostriatal pathways with distinct loops involving different cortical regions, which may be investigated using fMRI at an ultra-high field (7 T) with enhanced sensitivity compared to lower fields. OBJECTIVES: We performed fMRI at 7 T to assess functional connectivity between the striatum and several chosen cortical areas including the motor and prefrontal cortex, in order to better understand brain changes in the striatum-cortical pathways. METHOD: 13 manifest subjects (age 51 ± 13 years, cytosine-adenine-guanine [CAG] repeat 45 ± 5, Unified Huntington's Disease Rating Scale [UHDRS] motor score 32 ± 17), 8 subjects in the close-to-onset premanifest period (age 38 ± 10 years, CAG repeat 44 ± 2, UHDRS motor score 8 ± 2), 11 subjects in the far-from-onset premanifest period (age 38 ± 11 years, CAG repeat 42 ± 2, UHDRS motor score 1 ± 2), and 16 healthy controls (age 44 ± 15 years) were studied. The functional connectivity between the striatum and several cortical areas was measured by resting state fMRI at 7 T and analyzed in all participants. RESULTS: Compared to controls, functional connectivity between striatum and premotor area, supplementary motor area, inferior frontal as well as middle frontal regions was altered in HD (all p values <0.001). Specifically, decreased striatum-motor connectivity but increased striatum-prefrontal connectivity were found in premanifest HD subjects. Altered functional connectivity correlated consistently with genetic burden, but not with clinical scores. CONCLUSIONS: Differential changes in functional connectivity of striatum-prefrontal and striatum-motor circuits can be found in early and premanifest HD. This may imply a compensatory mechanism, where additional cortical regions are recruited to subserve functions that have been impaired due to HD pathology. Our results suggest the potential value of functional connectivity as a marker for future clinical trials in HD.