Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia.

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

X-linked hypophosphatemia: The medical expert's challenges and the patient's concerns on their journey with the disease.

X-linked hypophosphatemia (XLH) is a rare inheritable disorder of phosphate handling due to loss of function mutations of the PHEX gene, associated with increased production of FGF23 and impaired bone mineralization. In children, the disease's most common manifestations are bowing deformities of the lower limbs, short stature, and spontaneous dental abscesses. In adults, these are osteomalacia, insufficiency fractures, and enthesopathies associated with bone and joint pain. The XLH patient's journey with the disease may be difficult, reflecting concerns and experiences globally common to all patients with rare genetic diseases. Delays in diagnosis often preclude an optimal treatment outcome. Under-treatment is common as treating physicians, particularly those not familiar with the disease, tend to err on the side of caution, often choosing safety over efficacy. Physical abnormalities, pain, diminished function, and impaired mobility tend not only to isolate the XLH patient from his peers but also to have a significant psychological effect, eventually leading to significant impairment in quality of life. Significant advances in understanding the pathophysiology of XLH, the availability of a very comprehensive Evidence-based Guideline for the diagnosis and management of XLH, and the successful development of an effective and safe disease-specific novel therapy for XLH, have paved the way for a significant improvement in the management of this rare disorder of phosphate metabolism, heralding a significant improvement in the disease's outcome measures. Additional data from long-term observational studies and randomized controlled trials are eagerly awaited to consolidate these promising developments in the field of this rare disease.

Diagnosis and management of pseudohypoparathyroidism and related disorders: first international Consensus Statement.

This Consensus Statement covers recommendations for the diagnosis and management of patients with pseudohypoparathyroidism (PHP) and related disorders, which comprise metabolic disorders characterized by physical findings that variably include short bones, short stature, a stocky build, early-onset obesity and ectopic ossifications, as well as endocrine defects that often include resistance to parathyroid hormone (PTH) and TSH. The presentation and severity of PHP and its related disorders vary between affected individuals with considerable clinical and molecular overlap between the different types. A specific diagnosis is often delayed owing to lack of recognition of the syndrome and associated features. The participants in this Consensus Statement agreed that the diagnosis of PHP should be based on major criteria, including resistance to PTH, ectopic ossifications, brachydactyly and early-onset obesity. The clinical and laboratory diagnosis should be confirmed by a molecular genetic analysis. Patients should be screened at diagnosis and during follow-up for specific features, such as PTH resistance, TSH resistance, growth hormone deficiency, hypogonadism, skeletal deformities, oral health, weight gain, glucose intolerance or type 2 diabetes mellitus, and hypertension, as well as subcutaneous and/or deeper ectopic ossifications and neurocognitive impairment. Overall, a coordinated and multidisciplinary approach from infancy through adulthood, including a transition programme, should help us to improve the care of patients affected by these disorders.

Genetic and Epigenetic Defects at the GNAS Locus Lead to Distinct Patterns of Skeletal Growth but Similar Early-Onset Obesity.

Pseudohypoparathyroidism type 1A (PHP1A), pseudoPHP (PPHP), and PHP type 1B (PHP1B) are caused by maternal and paternal GNAS mutations and abnormal methylation at maternal GNAS promoter(s), respectively. Adult PHP1A patients are reportedly obese and short, whereas most PPHP patients are born small. In addition to parathyroid hormone (PTH) resistance, PHP1A and PHP1B patients may display early-onset obesity. Because early-onset and severe obesity and short stature are daily burdens for PHP1A patients, we aimed at improving knowledge on the contribution of the GNAS transcripts to fetal and postnatal growth and fat storage. Through an international collaboration, we collected growth and weight data from birth until adulthood for 306 PHP1A/PPHP and 220 PHP1B patients. PHP1A/PPHP patients were smaller at birth than healthy controls, especially PPHP (length Z-score: PHP1A -1.1 ± 1.8; PPHP -3.0 ± 1.5). Short stature is observed in 64% and 59% of adult PHP1A and PPHP patients. PHP1B patients displayed early postnatal overgrowth (height Z-score at 1 year: 2.2 ± 1.3 and 1.3 ± 1.5 in autosomal dominant and sporadic PHP1B) followed by a gradual decrease in growth velocity resulting in normal adult height (Z-score for both: -0.4 ± 1.1). Early-onset obesity characterizes GNAS alterations and is associated with significant overweight and obesity in adults (bodey mass index [BMI] Z-score: 1.4 ± 2.6, 2.1 ± 2.0, and 1.4 ± 1.9 in PPHP, PHP1A, and PHP1B, respectively), indicating that reduced Gsα expression is a contributing factor. The growth impairment in PHP1A/PPHP may be due to Gsα haploinsufficiency in the growth plates; the paternal XLαs transcript likely contributes to prenatal growth; for all disease variants, a reduced pubertal growth spurt may be due to accelerated growth plate closure. Consequently, early diagnosis and close follow-up is needed in patients with GNAS defects to screen and intervene in case of early-onset obesity and decreased growth velocity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

Progressive Development of PTH Resistance in Patients With Inactivating Mutations on the Maternal Allele of GNAS.

Context: Parathormone (PTH) resistance is characterized by hypocalcaemia, hyperphosphatemia, and elevated PTH in the absence of vitamin D deficiency. Pseudohypoparathyroidism type 1A [PHP1A, or inactivating parathormone (PTH)/PTHrp signaling disorder 2, according to the new classification (iPPSD2)], is caused by mutations in the maternal GNAS allele. Objective: To assess PTH resistance over time in 20 patients affected by iPPSD2 (PHP1A), diagnosed because of family history, ectopic ossification, or short stature, and carrying a GNAS mutation. Methods: We gathered retrospective data for calcium, phosphate, thyrotropin (TSH), and PTH levels at regular intervals. PTH infusion testing (teriparatide) was performed in 1 patient. Results: Patients were diagnosed at a mean age of 3.9 years and had a mean follow-up of 2 years. TSH resistance was already present at diagnosis in all patients (TSH, 13.3 ± 9.0 mIU/L). Over time, PTH levels increased (179 to 306 pg/mL; P < 0.05), and calcium levels decreased (2.31 to 2.21 mmol/L; P < 0.05), but phosphate levels did not decrease with age as expected for healthy individuals. One patient born with ectopic ossifications showed an increase in cyclic adenosine monophosphate upon PTH infusion, similar to that of controls, at 7 months of age, but an impaired response at 4 years of age. Conclusions: In patients with iPPSD2 (PHP1A), PTH resistance and hypocalcemia develop over time. These findings highlight the importance of screening for maternal GNAS mutations in the presence of ectopic ossifications or family history, even in the absence of PTH resistance and hypocalcemia. The follow-up of these patients should include regular assessments of calcium, phosphate, and PTH levels.

The immunoglobulin-like superfamily member IGSF3 is a developmentally regulated protein that controls neuronal morphogenesis.

The establishment of a functional brain depends on the fine regulation and coordination of many processes, including neurogenesis, differentiation, dendritogenesis, axonogenesis, and synaptogenesis. Proteins of the immunoglobulin-like superfamily (IGSF) are major regulators during this sequence of events. Different members of this class of proteins play nonoverlapping functions at specific developmental time-points, as shown in particular by studies of the cerebellum. We have identified a member of the little studied EWI subfamily of IGSF, the protein IGSF3, as a membrane protein expressed in a neuron specific- and time-dependent manner during brain development. In the cerebellum, it is transiently found in membranes of differentiating granule cells, and is particularly concentrated at axon terminals. There it co-localizes with other IGSF proteins with well-known functions in cerebellar development: TAG-1 and L1. Functional analysis shows that IGSF3 controls the differentiation of granule cells, more precisely axonal growth and branching. Biochemical experiments demonstrate that, in the developing brain, IGSF3 is in a complex with the tetraspanin TSPAN7, a membrane protein mutated in several forms of X-linked intellectual disabilities. In cerebellar granule cells, TSPAN7 promotes axonal branching and the size of TSPAN7 clusters is increased by downregulation of IGSF3. Thus IGSF3 is a novel regulator of neuronal morphogenesis that might function through interactions with multiple partners including the tetraspanin TSPAN7. This developmentally regulated protein might thus be at the center of a new signaling pathway controlling brain development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 75-92, 2017.

The Importance of Networking in Pseudohypoparathyroidism: EuroPHP Network and Patient Support Associations.

Pseudohypoparathyroidism is a rare endocrine disorder with an estimated prevalence of 1/100,000. It is characterized by hypocalcemia and hyperphosphatemia in the absence of vitamin D deficiency or impaired renal function. Research studies during the last 20 years have led to the identification of the molecular underlying cause of the disease, the characterization of the clinical and biochemical characteristics and the observation of an overlap between genetic and clinical manifestations. The creation of networks both for specialists (including endocrinologists, pediatricians, dermatologists, geneticists, molecular biologists…) and patients support groups brings up the opportunity of research advance, synergism and common objectives for families and investigators, improving the quality of information about the disease and its outcome, that, at the end, will improve both the knowledge and life of the patients and their families.

From pseudohypoparathyroidism to inactivating PTH/PTHrP signalling disorder (iPPSD), a novel classification proposed by the EuroPHP network.

OBJECTIVE: Disorders caused by impairments in the parathyroid hormone (PTH) signalling pathway are historically classified under the term pseudohypoparathyroidism (PHP), which encompasses rare, related and highly heterogeneous diseases with demonstrated (epi)genetic causes. The actual classification is based on the presence or absence of specific clinical and biochemical signs together with an in vivo response to exogenous PTH and the results of an in vitro assay to measure Gsa protein activity. However, this classification disregards other related diseases such as acrodysostosis (ACRDYS) or progressive osseous heteroplasia (POH), as well as recent findings of clinical and genetic/epigenetic background of the different subtypes. Therefore, the EuroPHP network decided to develop a new classification that encompasses all disorders with impairments in PTH and/or PTHrP cAMP-mediated pathway. DESIGN AND METHODS: Extensive review of the literature was performed. Several meetings were organised to discuss about a new, more effective and accurate way to describe disorders caused by abnormalities of the PTH/PTHrP signalling pathway. RESULTS AND CONCLUSIONS: After determining the major and minor criteria to be considered for the diagnosis of these disorders, we proposed to group them under the term 'inactivating PTH/PTHrP signalling disorder' (iPPSD). This terminology: (i) defines the common mechanism responsible for all diseases; (ii) does not require a confirmed genetic defect; (iii) avoids ambiguous terms like 'pseudo' and (iv) eliminates the clinical or molecular overlap between diseases. We believe that the use of this nomenclature and classification will facilitate the development of rationale and comprehensive international guidelines for the diagnosis and treatment of iPPSDs.

Oligomeric amyloid-ß peptide affects the expression of genes involved in steroid and lipid metabolism in primary neurons.

Amyloid-ß peptide (Aß) is the principal component of plaques in the brains of patients with Alzheimer's disease (AD), and the most toxic form of Aß may be as soluble oligomers. We report here the results of a microarray study of gene expression profiles in primary mouse cortical neurons in response to oligomeric Aß(1-42). A major and unexpected finding was the down-regulation of genes involved in the biosynthesis of cholesterol and other steroids and lipids (such as Fdft1, Fdps, Idi1, Ldr, Mvd, Mvk, Nsdhl, Sc4mol), the expression of which was verified by quantitative real-time RT-PCR (qPCR). The ATP-binding cassette gene Abca1, which has a major role in cholesterol transport in brain and other tissues and has been genetically linked to AD, was notably up-regulated. The possible involvement of cholesterol and other lipids in Aß synthesis and action in Alzheimer's disease has been studied and debated extensively but remains unresolved. These new data suggest that Aß may influence steroid and lipid metabolism in neurons via multiple gene-expression changes.

Dynamic association of tau with neuronal membranes is regulated by phosphorylation.

Tau is an abundant cytosolic protein which regulates cytoskeletal stability by associating with microtubules in a phosphorylation-dependent manner. We have found a significant proportion of tau is located in the membrane fraction of rat cortical neurons and is dephosphorylated, at least at Tau-1 (Ser199/Ser202), AT8 (Ser199/Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Inhibition of tau kinases casein kinase 1 (CK1) or glycogen synthase kinase-3 decreased tau phosphorylation and significantly increased amounts of tau in the membrane fraction. Mutation of serine/threonine residues to glutamate to mimic phosphorylation in the N-terminal, but not C-terminal, region of tau prevented its membrane localization in transfected cells, demonstrating that the phosphorylation state of tau directly impacts its localization. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn also induced tau dephosphorylation but did not affect its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that trafficking of tau between the cytosol and the neuronal membrane is dynamically regulated by tau phosphorylation through a mechanism dependent on fyn expression.

CRMP2 hyperphosphorylation is characteristic of Alzheimer's disease and not a feature common to other neurodegenerative diseases.

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that regulates neurite outgrowth. It is phosphorylated by Cdk5 and GSK3, and these modifications are abnormally high in the brains of Alzheimer's disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AßPP and PSEN1, but not AßPP or tau alone, where it is detectable before the appearance of amyloid plaques and neurofibrillary tangles, suggesting it is an early event in AD pathogenesis. Here, we have extended these observations by showing that CRMP2 is not hyperphosphorylated in mice overexpressing mutated PSEN1 alone, or in cultured neurons treated with soluble, oligomeric Aß42 peptide. Similarly, CRMP2 phosphorylation was not increased in a mouse model of severe neurodegeneration (PMSC-1 knockout) or in cultured neurons subjected to neurotoxic concentrations of NMDA or staurosporine. Most interestingly, CRMP2 phosphorylation was not increased in frontal cortex from patients with frontotemporal lobar degeneration associated with mutations in MAPT or with Pick bodies. Together, these observations are consistent with the hypothesis that abnormal phosphorylation of CRMP2 is specific to AD and occurs downstream of excessive processing of AßPP, but that neither excessive Aß42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau.

Recent reports have demonstrated that interactions between the microtubule-associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to ß-amyloid (Aß) in models of Alzheimer's disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aß-induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src-homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213-216) motif in tau plays an important role in mediating the interaction of tau with Fyn-SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn-SH3, is influenced by Fyn-mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn-SH2-tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent-resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn-dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn-mediated synaptic dysfunction that occurs in response to elevated Aß levels in neurodegenerative disease.

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.

BACKGROUND: Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as first step in screening putative membrane raft components. Traditional methods of raft isolation employed the nonionic detergent Triton X100. However successful separation of raft from non-raft domains in cells is dependent on matching the detergent used for raft isolation to the specific tissue under investigation. RESULTS: We report here the isolation of membrane rafts from primary neuronal culture using a panel of different detergents that gave rise to membrane fractions that differed in respect to cholesterol and protein content. In addition, proteomic profiling of neuronal membrane rafts isolated with different detergents, Triton X100 and CHAPSO, revealed heterogeneity in their protein content. CONCLUSIONS: These data demonstrate that appropriate selection of detergent for raft isolation is an important consideration for investigating raft protein composition of cultured neurons.

Membrane-bound beta-amyloid oligomers are recruited into lipid rafts by a fyn-dependent mechanism.

Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

Presenilin 2 mutations alter cystatin C trafficking in mouse primary neurons.

Mutations in the presenilin genes account for the majority of familial Alzheimer disease (FAD) cases. In the present report we demonstrated that the FAD-linked presenilin 2 mutations (PS2 M239I and T122R) alter cystatin C trafficking in mouse primary neurons reducing secretion of its glycosylated form. These mutations showed a different impact on cystatin C: PS2 T122R had a much stronger effect determining a dramatic intracellular accumulation of cystatin C (native and glycosylated), followed by a reduction in the secretion of both forms. Several experimental evidences suggest that cystatin C exerts a protective role in the brain and favors stem cells proliferation. Confocal imaging showed that the effect of PS2 T122R mutation was a massive recruitment of cystatin C into the neuronal processes, in the presence of an intact cytoskeletal structure. The consequent reduction in the cystatin C extracellular levels might result in a failure of neuroregeneration. Understanding the interplay of PS2 and cystatin C in the pathogenesis of AD might highlight new therapeutic prospective.