Molecular Brain

Congenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive. Herein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation. Our results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons’ inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients.

The analysis of the contributions to synaptic plasticity and memory of cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB has recruited the efforts of many laboratories all over the world. These are six key steps in the molecular biological delineation of short-term memory and its conversion to long-term memory for both implicit (procedural) and explicit (declarative) memory. I here first trace the background for the clinical and behavioral studies of implicit memory that made a molecular biology of memory storage possible, and then detail the discovery and early history of these six molecular steps and their roles in explicit memory.

Math5-null mutation results in the loss of retinal ganglion cells (RGCs) and in a concurrent increase of amacrine and cone cells. However, it remains unclear whether there is a cell fate switch of Math5-lineage cells in the absence of Math5 and whether MATH5 cell-autonomously regulates the differentiation of the above retinal neurons. Here, we performed a lineage analysis of Math5-expressing cells in developing mouse retinas using a conditional GFP reporter (Z/EG) activated by a Math5-Cre knock-in allele. We show that during normal retinogenesis, Math5-lineage cells mostly develop into RGCs, horizontal cells, cone photoreceptors, rod photoreceptors, and amacrine cells. Interestingly, amacrine cells of Math5-lineage cells are predominately of GABAergic, cholinergic, and A2 subtypes, indicating that Math5 plays a role in amacrine subtype specification. In the absence of Math5, more Math5-lineage cells undergo cell fate conversion from RGCs to the above retinal cell subtypes, and occasionally to cone-bipolar cells and Müller cells. This change in cell fate choices is accompanied by an up-regulation of NEUROD1, RXRγ and BHLHB5, the transcription factors essential for the differentiation of retinal cells other than RGCs. Additionally, loss of Math5 causes the failure of early progenitors to exit cell cycle and leads to a significant increase of Math5-lineage cells remaining in cell cycle. Collectively, these data suggest that Math5 regulates the generation of multiple retinal cell types via different mechanisms during retinogenesis.

Rối loạn nhận thức thần kinh liên quan đến HIV (HAND) được đặc trưng bởi sự phát triển của những bất thường về nhận thức, hành vi và vận động, xảy ra ở khoảng 50% cá nhân nhiễm HIV. Tại Hoa Kỳ, tỷ lệ hút thuốc lá dao động từ 35-70% ở các cá nhân nhiễm HIV so với 20% trong tổng dân số. Suy giảm nhận thức ở những người nghiện thuốc lá nặng đã được báo cáo rõ ràng. Tuy nhiên, các tác động cộng hưởng giữa nicotine và nhiễm HIV cũng như các cơ chế tiềm ẩn trong sự phát triển của HAND vẫn chưa được biết đến. Trong nghiên cứu này, chúng tôi đã khám phá vai trò của nicotine trong sự tiến triển của HAND bằng cách sử dụng dòng tế bào thần kinh SK-N-MC. Các tế bào SK-N-MC đã được nhiễm HIV-1 trong sự hiện diện hoặc vắng mặt của nicotine trong 7 ngày. Chúng tôi đã quan sát thấy sự gia tăng đáng kể khả năng nhiễm HIV ở SK-N-MC được điều trị bằng nicotine so với các tế bào thần kinh nhiễm HIV không điều trị. HIV và nicotine cộng hưởng để làm rối loạn đáng kể biểu hiện của các gen tính linh hoạt synapse và mật độ gai; đồng thời làm gia tăng mức độ HDAC2 trong tế bào SK-N-MC. Ngoài ra, việc ức chế sự gia tăng HDAC2 bằng cách sử dụng vorinostat dẫn đến sự phá vỡ tình trạng tiềm tàng của HIV và phục hồi biểu hiện của các gen tính linh hoạt synapse cũng như mật độ gai ở tế bào SK-N-MC bị nhiễm nicotine/HIV riêng lẻ và trong điều trị đồng thời. Hơn nữa, sự phosphoryl hóa gia tăng của eIF2 alpha, điều chỉnh tiêu cực quá trình dịch mã eukaryote, đã được quan sát thấy ở HIV đơn lẻ và trong điều trị đồng thời với nicotine so với tế bào SK-N-MC không điều trị và chỉ được điều trị bằng nicotine. Những kết quả này gợi ý rằng nicotine và HIV cộng hưởng để điều chỉnh tiêu cực sự biểu hiện gen tính linh hoạt synapse và mật độ gai, và điều này có thể góp phần vào việc tăng nguy cơ HAND ở những người hút thuốc nhiễm HIV. Ngoài việc làm rối loạn tình trạng tiềm tàng, vorinostat có thể là một liệu pháp có giá trị để ức chế các tác động điều chỉnh tiêu cực lên tính linh hoạt synapse ở những người nghiện nicotine nhiễm HIV.

Since 2009, evidence has accumulated to suggest that Tau aggregates form first in a small number of brain cells, from where they propagate to other regions, resulting in neurodegeneration and disease. Propagation of Tau aggregates is often called prion-like, which refers to the capacity of an assembled protein to induce the same abnormal conformation in a protein of the same kind, initiating a self-amplifying cascade. In addition, prion-like encompasses the release of protein aggregates from brain cells and their uptake by neighbouring cells. In mice, the intracerebral injection of Tau inclusions induced the ordered assembly of monomeric Tau, followed by its spreading to distant brain regions. Short fibrils constituted the major species of seed-competent Tau. The existence of several human Tauopathies with distinct fibril morphologies has led to the suggestion that different molecular conformers (or strains) of aggregated Tau exist.

In most animals, the nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS), the latter of which connects the CNS to all parts of the body. Damage and/or malfunction of the nervous system causes serious pathologies, including neurodegenerative disorders, spinal cord injury, and Alzheimer’s disease. Thus, not surprising, considerable research effort, both in vivo and in vitro, has been devoted to studying the nervous system and signal transmission through it. However, conventional in vitro cell culture systems do not enable control over diverse aspects of the neural microenvironment. Moreover, formation of certain nervous system growth patterns in vitro remains a challenge. In this study, we developed a deep hemispherical, microchannel-networked, concave array system and applied it to generate three-dimensional nerve-like neural bundles. The deep hemicylindrical channel network was easily fabricated by exploiting the meniscus induced by the surface tension of a liquid poly(dimethylsiloxane) (PDMS) prepolymer. Neurospheroids spontaneously aggregated in each deep concave microwell and were networked to neighboring spheroids through the deep hemicylindrical channel. Notably, two types of satellite spheroids also formed in deep hemispherical microchannels through self-aggregation and acted as an anchoring point to enhance formation of nerve-like networks with neighboring spheroids. During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells. These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids. Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca2+ signals in responding cells.

Many genetic and environmental factors are involved in the etiology of nicotine dependence. Although several candidate gene variations have been reported by candidate gene studies or genome-wide association studies (GWASs) to be associated with smoking behavior and the vulnerability to nicotine dependence, such studies have been mostly conducted with subjects with European ancestry. However, genetic factors have rarely been investigated for the Japanese population as GWASs. To elucidate genetic factors involved in nicotine dependence in Japanese, the present study comprehensively explored genetic contributors to nicotine dependence by using whole-genome genotyping arrays with more than 200,000 markers in Japanese subjects. The subjects for the GWAS and replication study were 148 and 374 patients, respectively. A two-stage GWAS was conducted using the Fagerström Test for Nicotine Dependence (FTND), Tobacco Dependence Screener (TDS), and number of cigarettes smoked per day (CPD) as indices of nicotine dependence. For the additional association analyses, patients who underwent major abdominal surgery, patients with methamphetamine dependence/psychosis, and healthy subjects with schizotypal personality trait data were recruited. Autopsy specimens with various diseases were also evaluated. After the study of associations between more than 200,000 marker single-nucleotide polymorphisms (SNPs) and the FTND, TDS, and CPD, the nonsynonymous rs2653349 SNP (located on the gene that encodes orexin [hypocretin] receptor 2) was selected as the most notable SNP associated with FTND, with a p value of 0.0005921 in the two-stage GWAS. This possible association was replicated for the remaining 374 samples. This SNP was also associated with postoperative pain, the initiation of methamphetamine use, schizotypal personality traits, and susceptibility to goiter. Although the p value did not reach a conventional genome-wide level of significance in our two-stage GWAS, we obtained significant results in the subsequent analyses that suggest that the rs2653349 SNP (Val308Ile) could be a genetic factor that is related to nicotine dependence and possibly pain, schizotypal personality traits, and goiter in the Japanese population.

The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer’s disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.

Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.