Proteome changes in autosomal recessive primary microcephaly.

BACKGROUND/AIM: Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. MATERIAL AND METHODS: We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. RESULT: We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. CONCLUSION: Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3.

PTRH2 is Necessary for Purkinje Cell Differentiation and Survival and its Loss Recapitulates Progressive Cerebellar Atrophy and Ataxia Seen in IMNEPD Patients.

Hom ozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2-/- mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2-/- knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease.

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability.

BACKGROUND: Minichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells. METHODS: Whole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of Mcm7 was characterised by performing quantitative real-time PCR, in situ hybridisation and immunostaining. To prove the disease-causative nature of identified MCM7, a proof-of-principle experiment was performed. RESULTS: We reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in MCM7 was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of Mcm7 in mice and a proliferative state: Mcm7 expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of Mcm7 in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant MCM7. CONCLUSION: We report mutations of MCM7 as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development.

The murine ortholog of Kaufman oculocerebrofacial syndrome protein Ube3b regulates synapse number by ubiquitinating Ppp3cc.

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.

Abnormal brain structure and behavior in MyD88-deficient mice.

While the original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis, the role of its ortholog Toll-like receptors (TLRs), the interleukin 1 receptor (IL-1R) family, and the associated signaling pathways in mammalian brain development and structure is poorly understood. Because the adaptor protein myeloid differentiation primary response protein 88 (MyD88) is essential for downstream signaling of most TLRs and IL-1R, we systematically investigated the effect of MyD88 deficiency on murine brain structure during development and on behavior. In neonatal Myd88-/- mice, neocortical thickness was reduced, while density of cortical neurons was increased. In contrast, microglia, astrocyte, oligodendrocyte, and proliferating cell numbers were unchanged in these mice compared to wild-type mice. In adult Myd88-/- mice, neocortical thickness was unaltered, but neuronal density in neocortex and hippocampus was increased. Neuron arborization was less pronounced in adult Myd88-/- mice compared to wild-type animals. In addition, numbers of microglia and proliferating cells were increased in the neocortex and subventricular zone, respectively, with unaltered astrocyte and oligodendrocyte numbers, and myelinization was enhanced in the adult Myd88-/- neocortex. These morphologic changes in the brain of adult Myd88-/- mice were accompanied by specific behavioral traits, such as decreased locomotor activity, increased anxiety-like behavior, but normal day/light activity, satisfactory learning, short- and long-term spatial memory, potential cognitive inflexibility, and increased hanging and locomotor behavior within their home cage. Taken together, MyD88 deficiency results in morphologic and cellular changes in the mouse brain, as well as in altered natural and specific behaviors. Our data indicate a pathophysiological significance of MyD88 for mammalian CNS development, structure, and function.

Teaching Musculoskeletal Module using dissection videos: feedback from medical students.

BACKGROUND AND AIMS: Over the last two decades many medical schools have been exploring alternatives to hands-on cadaver dissection in teaching anatomy. This study aimed at reporting medical students' feedback on using dissection videos in teaching anatomy of the musculoskeletal system. METHODS: Dissection videos were used to teach the anatomy of the musculoskeletal system for third year medical students. At the end of the module, feedbacks from medical students were reported using a questionnaire designed for this purpose. Statistically valid responses were considered for 284 students. RESULTS: Around 60% of the students enjoyed learning anatomy by watching dissection videos but the majority - mostly non-Jordanian - thought that the duration of the videos should be shorter. 83% (236/284)of the students enjoyed the presence of an instructor to guide them through the video and 85% (241/284) wanted to discuss the content with the instructor after watching. Most of the students liked to have access to the videos at any time in an open lab policy. Only 23% (66/284) of the students - mostly Jordanian - were willing to completely replace cadaveric prosections with dissection videos. Most of the students found that dissection videos helped them to understand anatomy lectures in a better way and in memorizing anatomical details. A significantly higher percentage of Jordanian students preferred watching dissection videos at home and preferred dissection videos to replace traditional anatomy lab sessions. CONCLUSIONS: In the light of our present findings, using dissection videos as a teaching method of anatomy was well received by students. However, it seemed that the students wanted dissection videos to be integrated with using cadaveric prosections rather than replacing them.

Congenital microcephaly-linked CDK5RAP2 affects eye development.

Biallelic mutations in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause autosomal recessive primary microcephaly type 3 (MCPH3). MCPH is characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. Only recently, congenital cataracts were reported in four patients of one pedigree with MCPH3. Given the lack of a further pedigree with this phenotype, it remained unclear whether this was a true causal relationship. Here we support the link between CDK5RAP2 and eye development by showing that most Cdk5rap2 mutant mice (an/an) exhibit eye malformations ranging from reduced size of one or both eyes (microphthalmia) to total absence of both eyes (anophthalmia). We also detected increased apoptosis in the an/an retinal progenitor cells associated with more mitotic cells. This indicates an important role of Cdk5rap2 in physiologic eye development.

Altered inhibition and excitation in neocortical circuits in congenital microcephaly.

Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity.

Autosomal Recessive Primary Microcephaly (MCPH): An Update.

Autosomal recessive primary microcephaly (MCPH; MicroCephaly Primary Hereditary) is a genetically heterogeneous neurodevelopmental disorder characterized by a significantly reduced head circumference present already at birth and intellectual disability. Inconsistent features include hyperactivity, an expressive speech disorder, and epilepsy. Here, we provide a brief overview on this rare disorder pertinent for clinicians.

Neuronal Cell Types in the Spinal Trigeminal Nucleus of the Camel Brain.

Neurons in the spinal trigeminal nucleus of a camel were morphologically studied by the Golgi impregnation method. The neurons were classified based on the size and shape of their cell bodies, the density of their dendritic trees, and the morphology and distribution of their appendages. At least 12 morphological types of neurons were found in the camel spinal trigeminal nucleus, including the following: stalked, islets, octopus-like, lobulated, boat-like, pyramidal, multipolar, round, oval, and elongated neurons. These neurons exhibited large numbers of various forms of appendages that arise not only from their dendrites but also from their cell bodies. Moreover, neurons with unique large dilatations especially at their dendritic branching points were also reported. The neurons reported in this study displayed an array of different sizes and shapes and featured various forms of appendages arising from cell bodies and dendrites. Such morphologically distinctive neuronal cell types might indicate an evolutionary adaptation to pain and temperature processing pathways at the level of the spinal trigeminal nucleus in camels, which traditionally live in a very harsh climatic environment and are frequently exposed to painful stimuli.

A Golgi study of neurons in the camel cerebellum (Camelus dromedarius).

Neurons in the cerebellar cortex of camels were studied using modified Golgi impregnation methods. Neurons were classified according to their position, morphology of their soma, density and distribution of dendrites, and the course of their axons. Accordingly, eight types of neurons were identified. Three types were found in the molecular layer: upper and lower stellate cells and basket cells, and four types were found in the granular layer: granule cells, Golgi Type II cells, Lugaro cells, and unipolar brush cells. Only the somata of Purkinje cells were found in the Purkinje cell layer. The molecular layer is characterized by the presence of more dendrites, dendritic spines, and transverse fibers. Golgi cells also show extensive dendritic branching and spines. The results illustrate the neuronal features of the camel cerebellum as a large mammal living in harsh environmental conditions. These findings should contribute to advancing our understanding of species-comparative anatomy in achieving better coordination of motor activity.

Autosomal Recessive Primary Microcephaly: Not Just a Small Brain.

Microcephaly or reduced head circumference results from a multitude of abnormal developmental processes affecting brain growth and/or leading to brain atrophy. Autosomal recessive primary microcephaly (MCPH) is the prototype of isolated primary (congenital) microcephaly, affecting predominantly the cerebral cortex. For MCPH, an accelerating number of mutated genes emerge annually, and they are involved in crucial steps of neurogenesis. In this review article, we provide a deeper look into the microcephalic MCPH brain. We explore cytoarchitecture focusing on the cerebral cortex and discuss diverse processes occurring at the level of neural progenitors, early generated and mature neurons, and glial cells. We aim to thereby give an overview of current knowledge in MCPH phenotype and normal brain growth.

Neuronal cell types in the anterior ventral thalamic nucleus of the camel.

The anterior ventral nucleus neurons in of the camel brain were morphologically studied by Golgi impregnation method. Two neuronal types of were found in the camel anterior ventral thalamic nucleus, namely, Golgi-type I neurons and Golgi-type II neurons. Those neurons were generally similar to their counterparts in the human thalamus. The Golgi-type I neurons exhibited medium to large cell body (mean diameter = 25 µm) which was either multipolar or triangular in shape. They had from 3 to 10 primary dendrites with many branches but with no spines or appendages. The Golgi-type II neurons had small to medium size (somatic mean diameter = 17.5 µm), their cell bodies were variable in shape, some were round, and others were multipolar or fusiform. These cells bodies had two to six primary dendrites with few branches that may have spines and/or grape-like appendages. Our findings shed some light on the anterior ventral thalamic nucleus structure of the camel as one of the strongest adaptive mammals to the hard climatic conditions.

Immunofluorescence Staining of Paraffin Sections Step by Step.

Immunofluorescence staining is the most frequently applied technique to detect and visualize various molecules in biological samples. Many protocols can be found in the literature and the websites of commercial antibody producers. This can result in a time-consuming and costly methodical work to establish "simple" antibody staining. We here summarize in a stepwise manner an easy-to-follow immunofluorescence staining protocol with an improved specific fluorescent signal and a reduced background and non-specific binding signal. This will help scientists to save time, effort, and antibody costs during the application of such a valuable technique.

CDK5RAP2 Is Required to Maintain the Germ Cell Pool during Embryonic Development.

Gene products linked to microcephaly have been studied foremost for their role in brain development, while their function in the development of other organs has been largely neglected. Here, we report the critical role of Cdk5rap2 in maintaining the germ cell pool during embryonic development. We highlight that infertility in Cdk5rap2 mutant mice is secondary to a lack of spermatogenic cells in adult mice as a result of an early developmental defect in the germ cells through mitotic delay, prolonged cell cycle, and apoptosis.

Golgi-Cox Staining Step by Step.

Golgi staining remains a key method to study neuronal morphology in vivo. Since most protocols delineating modifications of the original staining method lack details on critical steps, establishing this method in a laboratory can be time-consuming and frustrating. Here, we describe the Golgi-Cox staining in such detail that should turn the staining into an easily feasible method for all scientists working in the neuroscience field.

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes.

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.

A Golgi study of the camel cuneate nucleus.

Neurons in the cuneate nucleus of the camel brain stem were studied by Golgi method. Six types of neurons were identified based on soma size and shape, density of dendritic trees, morphology and distribution of spines, and appendages. Type I neurons had large spherical somata with somatic appendages. Dendritic appendages were predominant on proximal dendrites with terminal flower-like appendages. Type II neurons had medium to large soma. Appendages and spines were found for the soma as well as along dendrites of different orders. Axons with local branches were seen for these neurons. Type III neurons were small to medium spheroidal or triangulated with large number of spines and appendages on all parts of neurons including soma, dendrites, and initial axonal segments. Axons of these neurons branch profusely and formed rich local axonal arborizations. Type IV medium-size neurons have bipolar, round, or fusiform soma with somatic spines. Their dendrites were sparsely branching with spines and terminal side branches. Type V neurons were spheroid or triangular with small soma with somatic appendages. Their dendrites were sparsely branching and terminate as thin spiny side branches. Type VI neurons were small-size unipolar, round, or fusiform with some dendritic spines and protrusions. These findings shed some light on the structure of the cuneate nucleus of one of the largest animals (the camel).