Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

BACKGROUND: Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). AIMS: Identification of genomic disorders in DD/ID. MATERIALS AND METHODS: We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. RESULTS: We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. DISCUSSION AND CONCLUSION: We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Rare structural variation of synapse and neurotransmission genes in autism.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

A proof-of-concept study of growth hormone in children with Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome (PMS) is caused by 22q13 deletions including SHANK3 or pathogenic sequence variants in SHANK3 and is among the more common rare genetic findings in autism spectrum disorder (ASD). SHANK3 is critical for synaptic function, and preclinical and clinical studies suggest that insulin-like growth factor-1 (IGF-1) can reverse a range of deficits in PMS. IGF-1 release is stimulated by growth hormone secretion from the anterior pituitary gland, and this study sought to assess the feasibility of increasing IGF-1 levels through recombinant human growth hormone (rhGH) treatment, in addition to establishing safety and exploring efficacy of rhGH in children with PMS. METHODS: rhGH was administered once daily for 12 weeks to six children with PMS using an open-label design. IGF-1 levels, safety, and efficacy assessments were measured every 4 weeks throughout the study. RESULTS: rhGH administration increased levels of IGF-1 by at least 2 standard deviations and was well tolerated without serious adverse events. rhGH treatment was also associated with clinical improvement in social withdrawal, hyperactivity, and sensory symptoms. LIMITATIONS: Results should be interpreted with caution given the small sample size and lack of a placebo control. CONCLUSIONS: Overall, findings are promising and indicate the need for larger studies with rhGH in PMS. Trial registration NCT04003207. Registered July 1, 2019, https://clinicaltrials.gov/ct2/show/NCT04003207 .

Clinical trial of insulin-like growth factor-1 in Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome (PMS) is caused by haploinsufficiency of the SHANK3 gene and is characterized by global developmental delays and autism spectrum disorder (ASD). Based on several converging lines of preclinical and clinical evidence supporting the use of insulin-like growth factor-1 (IGF-1) in PMS, this study aims to follow-up a previous pilot study with IGF-1 to further evaluate this novel therapeutic for core symptoms of ASD in children with PMS. METHODS: Ten children aged 5-9 with PMS were enrolled. Participants were randomized to receive IGF-1 or placebo (saline) using a 12-week, double-blind, crossover design. Efficacy was assessed using the primary outcome of the Aberrant Behavior Checklist-Social Withdrawal (ABC-SW) subscale as well as secondary outcome measures reflecting core symptoms of ASD. To increase power and sample size, we jointly analyzed the effect of IGF-1 reported here together with results from our previous controlled trail of IGF-1 in children with PMS (combined N = 19). RESULTS: Results on the ABC-SW did not reach statistical significance, however significant improvements in sensory reactivity symptoms were observed. In our pooled analyses, IGF-1 treatment also led to significant improvements in repetitive behaviors and hyperactivity. There were no other statistically significant effects seen across other clinical outcome measures. IGF-1 was well tolerated and there were no serious adverse events. LIMITATIONS: The small sample size and expectancy bias due to relying on parent reported outcome measures may contribute to limitations in interpreting results. CONCLUSION: IGF-1 is efficacious in improving sensory reactivity symptoms, repetitive behaviors, and hyperactivity  in children with PMS. Trial registration NCT01525901.

High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.

Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment.

Most early-onset familial Alzheimer disease (AD) cases are caused by mutations in the highly related genes presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations produce increases in beta-amyloid (Abeta) formation and apoptosis in many experimental systems. A cDNA (ALG-3) encoding the last 103 amino acids of PS2 has been identified as a potent inhibitor of apoptosis. Using this PS2 domain in the yeast two-hybrid system, we have identified a neuronal protein that binds calcium and presenilin, which we call calsenilin. Calsenilin interacts with both PS1 and PS2 in cultured cells, and can regulate the levels of a proteolytic product of PS2. Thus, calsenilin may mediate the effects of wild-type and mutant presenilins on apoptosis and on Abeta formation. Further characterization of calsenilin may lead to an understanding of the normal role of the presenilins and of the role of the presenilins in Alzheimer disease.

Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides.

Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.

A randomized controlled trial of intranasal oxytocin in Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare neurodevelopmental disorder caused by haploinsufficiency of the SHANK3 gene and characterized by global developmental delays, deficits in speech and motor function, and autism spectrum disorder (ASD). Monogenic causes of ASD such as PMS are well suited to investigations with novel therapeutics, as interventions can be targeted based on established genetic etiology. While preclinical studies have demonstrated that the neuropeptide oxytocin can reverse electrophysiological, attentional, and social recognition memory deficits in Shank3-deficient rats, there have been no trials in individuals with PMS. The purpose of this study is to assess the efficacy and safety of intranasal oxytocin as a treatment for the core symptoms of ASD in a cohort of children with PMS. METHODS: Eighteen children aged 5-17 with PMS were enrolled. Participants were randomized to receive intranasal oxytocin or placebo (intranasal saline) and underwent treatment during a 12-week double-blind, parallel group phase, followed by a 12-week open-label extension phase during which all participants received oxytocin. Efficacy was assessed using the primary outcome of the Aberrant Behavior Checklist-Social Withdrawal (ABC-SW) subscale as well as a number of secondary outcome measures related to the core symptoms of ASD. Safety was monitored throughout the study period. RESULTS: There was no statistically significant improvement with oxytocin as compared to placebo on the ABC-SW (Mann-Whitney U = 50, p = 0.055), or on any secondary outcome measures, during either the double-blind or open-label phases. Oxytocin was generally well tolerated, and there were no serious adverse events. LIMITATIONS: The small sample size, potential challenges with drug administration, and expectancy bias due to relying on parent reported outcome measures may all contribute to limitations in interpreting results. CONCLUSION: Our results suggest that intranasal oxytocin is not efficacious in improving the core symptoms of ASD in children with PMS. Trial registration NCT02710084.

The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

SLC25A12 expression is associated with neurite outgrowth and is upregulated in the prefrontal cortex of autistic subjects.

Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.

No obvious abnormality in mice deficient in receptor protein tyrosine phosphatase beta.

The development of neurons and glia is governed by a multitude of extracellular signals that control protein tyrosine phosphorylation, a process regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Receptor PTPbeta (RPTPbeta; also known as PTPzeta) is expressed predominantly in the nervous system and exhibits structural features common to cell adhesion proteins, suggesting that this phosphatase participates in cell-cell communication. It has been proposed that the three isoforms of RPTPbeta play a role in regulation of neuronal migration, neurite outgrowth, and gliogenesis. To investigate the biological functions of this PTP, we have generated mice deficient in RPTPbeta. RPTPbeta-deficient mice are viable, are fertile, and showed no gross anatomical alterations in the nervous system or other organs. In contrast to results of in vitro experiments, our study demonstrates that RPTPbeta is not essential for neurite outgrowth and node formation in mice. The ultrastructure of nerves of the central nervous system in RPTPbeta-deficient mice suggests a fragility of myelin. However, conduction velocity was not altered in RPTPbeta-deficient mice. The normal development of neurons and glia in RPTPbeta-deficient mice demonstrates that RPTPbeta function is not necessary for these processes in vivo or that loss of RPTPbeta can be compensated for by other PTPs expressed in the nervous system.

Examining the Efficacy of a Family Peer Advocate Model for Black and Hispanic Caregivers of Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects individuals across all racial and ethnic groups, yet rates of diagnosis are disproportionately higher for Black and Hispanic children. Caregivers of children with ASD experience significant stressors, which have been associated with parental strain, inadequate utilization of mental health services and lower quality of life. The family peer advocate (FPA) model has been utilized across service delivery systems to provide family-to-family support, facilitate engagement, and increase access to care. This study used a randomized controlled design to examine the efficacy of FPAs in a racially and ethnically diverse sample. Results demonstrate significantly increased knowledge of ASD and reduced levels of stress for caregivers who received the FPA intervention as compared to treatment as usual.

The nature and metabolism of potentially amyloidogenic carboxyl-terminal fragments of the Alzheimer beta/A4-amyloid precursor protein: some technical notes.

The proteolytic processing and secretion of APP are regulated by protein phosphorylation, especially via protein kinase C and protein phosphatases 1 and/or 2A. Our studies of these regulatory mechanisms have led us to perform extensive experimentation on the metabolism of APP carboxyl-terminal fragments, using as our system either untransfected, undifferentiated rat pheochromocytoma (PC12) cells or APP-baculovirus infected Sf9 cells. We have not assayed APP fragments for biological activity in either system. However, we have made potentially relevant observations regarding APP carboxyl-terminal fragment trafficking. In this note, we review our published and unpublished data in relation to published reports from other laboratories using related systems.

Neuronal cyclooxygenase 2 expression in the hippocampal formation as a function of the clinical progression of Alzheimer disease.

BACKGROUND: Prior studies have shown that cyclooxygenase 2 (COX-2), an enzyme involved in inflammatory mechanisms and neuronal activities, is up-regulated in the brain with Alzheimer disease (AD) and may represent a therapeutic target for anti-inflammatory treatments. OBJECTIVE: To explore COX-2 expression in the brain as a function of clinical progression of early AD. DESIGN AND MAIN OUTCOME MEASURES: Using semiquantitative immunocytochemistry, we analyzed COX-2 protein content in the hippocampal formation in 54 postmortem brain specimens from patients with normal or impaired cognitive status. SETTING AND PATIENTS: Postmortem study of nursing home residents. RESULTS: The immunointensity of COX-2 signal in the CA3 and CA2 but not CA1 subdivisions of the pyramidal layers of the hippocampal formation of the AD brain increased as the disease progressed from questionable to mild clinical dementia as assessed by Clinical Dementia Rating. COX-2 signal was increased in all 3 regions examined among cases characterized by severe dementia. CONCLUSION: Neuronal COX-2 content in subsets of hippocampal pyramidal neurons may be an indicator of progression of dementia in early AD.

Correlation between Abetax-40-, Abetax-42-, and Abetax-43-containing amyloid plaques and cognitive decline.

CONTEXT: Accumulation of senile plaques containing amyloid beta (Abeta)-protein is a pathologic hallmark of Alzheimer disease. Amyloid beta-peptide is heterogeneous, with carboxyterminal variants ending at residues Val40 (Abetax-40), Ala42 (Abetax-42), or Thr43 (Abetax-43). The relative importance of each of these variants in dementia or cognitive decline remains unclear. OBJECTIVE: To study whether Abeta deposition correlates with dementia and occurs at the earliest signs of cognitive decline. DESIGN, SETTING, AND PATIENTS: Postmortem cross-sectional study comparing the deposition of Abeta variants in the prefrontal cortex of 79 nursing home residents having no, questionable, mild, moderate, or severe dementia. MAIN OUTCOME MEASURES: Levels of staining of Abeta-peptides ending at amino acid 40, 42, or 43 in the frontal cortex, as a function of Clinical Dementia Rating score. RESULTS: There were significant deposits of all 3 Abeta species that strongly correlated with cognitive decline. Furthermore, deposition of Abetax-42 and Abetax-43 occurred very early in the disease process before there could be a diagnosis of Alzheimer disease. Levels of deposited Abetax-43 appeared surprisingly high given the low amounts synthesized. CONCLUSIONS: These data indicate that Abetax-42 and Abetax-43 are important species associated with early disease progression and suggest that the physiochemical properties of the Abeta species may be a major determinant in amyloid deposition. The results support an important role for Abeta in mediating initial pathogenic events in Alzheimer disease dementia and reinforce that treatment strategies targeting the formation, accumulation, or cytotoxic effects of Abeta should be pursued.

Biochemical and immunocytochemical characterization of calsenilin in mouse brain.

Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer's disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.

Subcellular localization of presenilin 2 endoproteolytic C-terminal fragments.

Mutations in the genes that encode the presenilin 1 and 2 (PS1 and PS2) proteins cause the majority of familial Alzheimer's disease (FAD). Differential cleavage of the presenilins results in a generation of at least two C-terminal fragments (CTFs). An increase in the smaller of these two CTFs is one of the few changes in presenilin processing associated with FAD mutations in both PS1 and PS2. Interestingly, the phosphorylation of PS2 modulates the production of the smaller, caspase-derived PS2 CTF, which indicates that the generation of this fragment is a regulated, physiologic event. To date, there is no data concerning the subcellular distribution of the caspase-derived PS2 CTF. Because this fragment is normally present at levels that are difficult to detect, we have used cell lines in which the production of wild-type or N141I mutant PS2 is controlled by a tetracycline-regulated promoter in order to assess the subcellular localization of the caspase CTF in relation to the larger, constitutive PS2 CTF and to PS2 holoprotein. We have found that when levels of PS2 are low, the constitutive CTF colocalizes with markers consistent with localization in the early Golgi-ER-Golgi intermediate compartment (ERGIC) while the caspase CTF colocalizes with markers for the endoplasmic reticulum (ER). Following induction of wild-type or mutant PS2, when the levels of PS2 are high, the primary localization of the constitutive CTF appears to shift from the early Golgi-ERGIC in addition to the ER. Interestingly, while the induction of wild-type PS2 resulted in the localization of the caspase CTF primarily in the ER, the induction of mutant PS2 resulted in the localization of the caspase CTF to both the ER and the early Golgi-ERGIC. In summary, these data suggest that the two presenilin 2 CTFs have different patterns of subcellular localization and that the N141I PS2 mutation alters the localization pattern of the PS2 caspase fragment.

Regulation of APP processing by intra- and intercellular signals.

APP processing appears to be under complex regulation. This regulation is apparently important under both normal and pathological conditions. Of direct clinical interest is the observation that A beta formation can be regulated by various means. This raises the possibility that altered APP processing may cause an increase in A beta formation in AD, and suggests that it may be possible to regulate the production of A beta as a therapeutic approach in AD. As an example of the utility of the latter approach, consider a patient carrying the Swedish APP mutation. If it is true that the cause of AD in such a patient is due to increased A beta production, then decreasing A beta production should delay the onset of the disease. Even in individuals where increased A beta formation is not the cause of AD but there is some other causes, such as the presence of an allele of apolipoprotein E which causes A beta accumulation and hence synaptic loss, decreasing A beta formation may be beneficial. It is of course a very long way from in vitro experiments to therapy. The current emphasis on studying APP processing in vivo represents the next step towards this goal.