Ulk2 controls cortical excitatory-inhibitory balance via autophagic regulation of p62 and GABAA receptor trafficking in pyramidal neurons.

Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.

A noncompetitive BACE1 inhibitor TAK-070 ameliorates Abeta pathology and behavioral deficits in a mouse model of Alzheimer's disease.

We discovered a nonpeptidic compound, TAK-070, that inhibited BACE1, a rate-limiting protease for the generation of Abeta peptides that are considered causative for Alzheimer's disease (AD), in a noncompetitive manner. TAK-070 bound to full-length BACE1, but not to truncated BACE1 lacking the transmembrane domain. Short-term oral administration of TAK-070 decreased the brain levels of soluble Abeta, increased that of neurotrophic sAPPalpha by approximately 20%, and normalized the behavioral impairments in cognitive tests in Tg2576 mice, an APP transgenic mouse model of AD. Six-month chronic treatment decreased cerebral Abeta deposition by approximately 60%, preserving the pharmacological efficacy on soluble Abeta and sAPPalpha levels. These results support the feasibility of BACE1 inhibition with a noncompetitive inhibitor as disease-modifying as well as symptomatic therapy for AD.

A nonsense mutation in Gnat1, encoding the alpha subunit of rod transducin, in spontaneous mouse models of retinal dysfunction.

ICR-derived retinal dysfunction (IRD) 1 and IRD2 mice are new spontaneous mouse models of rod-cone and rod dysfunctions, respectively. In this study, we investigated the cause of rod dysfunction in IRD1 and IRD2 mice. Gene expression of rod phototransduction proteins was analyzed by quantitative real-time RT-PCR. mRNA levels of Gnat1, which encodes the alpha subunit of rod transducin (Tralpha), were severely reduced. Tralpha protein was immunohistochemically undetectable in both IRD1 and IRD2 mice. Sequencing of Tralpha cDNA revealed a 48-base pair (bp) insertion between exons 4 and 5 in both mutant strains. The insertion changed codon 150 (TAC) to a stop codon (TAG) (Tyr150Ter). The truncated Tralpha protein was undetectable in the retinas of both mutants by western blot analysis using a primary antibody against the N-terminal region. A 57-bp deletion was identified in intron 4 of the Gnat1 gene, which encodes the Tralpha protein, and included the last two bases of the splice donor site of intron 4. Thus our results showed that IRD1 and IRD2 mice harbor a nonsense mutation in the Gnat1 gene, resulting in the absence or suppressed expression of the Tralpha protein, which is the likely cause of rod dysfunction in both mutants.

A mouse model of 22q11.2 deletions: Molecular and behavioral signatures of Parkinson's disease and schizophrenia.

Individuals with chromosome 22q11.2 deletions are at increased risk of developing psychiatric conditions, most notably, schizophrenia (SZ). Recently, clinical studies have also implicated these recurrent 22q11.2 deletions with the risk of early-onset Parkinson's disease (PD). Thus far, the multiple mouse models generated for 22q11.2 deletions have been studied primarily in the context of congenital cardiac, neurodevelopmental, and psychotic disorders. One of these is the Df1/+ model, in which SZ-associated and developmental abnormalities have been reported. We present the first evidence that the mouse model for the 22q11.2 deletion exhibits motor coordination deficits and molecular signatures (that is, elevated α-synuclein expression) relevant to PD. Reducing the α-synuclein gene dosage in Df1/+ mice ameliorated the motor deficits. Thus, this model of the 22q11.2 deletion shows signatures of both SZ and PD at the molecular and behavioral levels. In addition, both SZ-associated and PD-relevant deficits in the model were ameliorated by treatment with a rapamycin analog, CCI-779. We now posit the utility of 22q11.2 deletion mouse models in investigating the mechanisms of SZ- and PD-associated manifestations that could shed light on possible common pathways of these neuropsychiatric disorders.

Methylphenidate and Guanfacine Ameliorate ADHD-Like Phenotypes in Fez1-Deficient Mice.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that, while prevalent, has a stagnant track record for advances in treatment. The limited availability of animal models with appropriate face and predictive validities has hampered progress in developing novel neurobiological hypotheses and testing new therapeutic options for this condition. Here, we report that mice deficient in Fez1, a gene specifically expressed in the nervous system with documented functions in neurodevelopment, show hyperactivity and impulsivity phenotypes, which are ameliorated by administering methylphenidate (MPH) or guanfacine (GFC), two pharmacological agents used for ADHD treatment. Fez1-knockout (KO) mice show reduced expression of tyrosine hydroxylase in the midbrain and the brain stem and have reduced levels of dopamine, norepinephrine, or their metabolites in both the nucleus accumbens and the prefrontal cortex. These neurochemical changes in Fez1-KO mice were normalized by MPH or GFC. We propose that Fez1-KO mice can be used as a model to evaluate the role of altered neurodevelopment in the manifestation of ADHD-like behavioral phenotypes, as well as to investigate the neurobiological mechanisms of existing and new pharmacotherapeutic agents for ADHD.

Ulk1 protects against ethanol-induced neuronal stress and cognition-related behavioral deficits.

Alcoholism is a psychiatric condition that develops through neuroadaptations in response to neuronal stresses caused by chronic ethanol intake. Neurons can adapt to ethanol-induced metabolic changes by activating cellular protective mechanisms, including autophagy. Here we show that expression of Ulk1, a gene critical to the regulation of autophagy, was affected in the prefrontal cortex (PFC) of mice following chronic intermittent ethanol (CIE) exposure. Consequently, overall levels of Ulk1 activity in the PFC were downregulated, leading to accumulation of p62, a protein that serves as a target for autophagic degradation. In addition, Ulk1-null mice demonstrated decline in the exploratory activity, deficits in the ability to recognize novel objects following CIE exposure, and reduced rate of voluntary ethanol drinking. The data suggest the neuroprotective role for Ulk1-mediated autophagy in the suppression of neuropsychiatric manifestation during ethanol exposure.

[Reduction in n-3 PUFA levels and EPA/AA ratio in serum after desert travels in China].

OBJECTIVES: The effects of 3 months of desert travel in China on serum fatty acids and tocopherol were studied. METHODS: In project staff members (6 males, 3 females, aged 19-27 years), serum levels of fatty acids and alpha-tocopherol were analyzed before and after travel by gas liquid chromatography and high-performance liquid chromatography, respectively. RESULTS: Comparison of the levels before and after the trip showed no differences in serum total cholesterol, triglycerides, total protein or alpha-tocopherol. There were no changes in the levels of total fatty acids, while the percentage of polyunsaturated fatty acids increased (p < 0.05). Levels of n-3 PUFA lowered from 166 micrograms/ml to 103 micrograms/ml, and those of n-6 PUFA had increased from 988 micrograms/ml to 1140 micrograms/ml after the trip (p < 0.01 and p < 0.001, respectively). No change was observed in the serum levels of alpha-linolenic acid (C18:3n-3), but lowering of the levels of eicosapentaenoic acid (EPA, C20:5n-3) from 41.4 micrograms/ml to 16.3 micrograms/ml and docosahexaenoic acid (DHA, C22:6n-3) from 107.8 micrograms/ml to 71.7 micrograms/ml was found after the trip (p < 0.05 and p < 0.01, respectively). Serum levels of linoleic acid (LA, C18:2n-6) increased from 832 micrograms/ml to 598 micrograms/ml (p < 0.001), and arachidonic acid (AA, C20:4n-6) tended to increase. The ratios of n-3/n-6 PUFA and EPA/AA decreased from 0.171 to 0.091 and from 0.258 to 0.096 after the trip, respectively (p < 0.01 for both). CONCLUSIONS: Our findings indicated that 3 months of desert travel increased the serum levels of n-6 PUFA and LA and reduced the serum levels of n-3 PUFA and EPA and the ratios of n-3/n-6 PUFA and EPA/AA, possibly due to a relative essential fatty acid deficiency.

Hypothermia enhances heat-shock protein 70 production in ischemic brains.

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Inverse relationship between serum erythropoietin and blood lead concentrations in Kathmandu tricycle taxi drivers.

OBJECTIVE: Kathmandu tricycle taxi drivers, whose environmental lead (Pb) exposure is ascribable mainly to vehicular exhaust, were studied to examine a dose-response relationship between blood Pb (Pb-B) and serum erythropoietin (sEPO) concentrations. METHODS: Subjects were 27 drivers and 9 non-drivers. They were non-anemic healthy men with normal renal function. Pb-B was measured by an atomic absorption spectrometer with a graphite furnace, and sEPO was determined with a sandwich-type enzyme-linked immunosorbent assay. RESULTS: sEPO levels in drivers were lower than those of non-drivers, while Pb-B levels in drivers were higher than those of non-drivers. There was an inverse relationship between Pb-B and sEPO. CONCLUSIONS: The data suggest that Pb inhibits renal EPO production in a dose-dependent manner in persons with subclinical Pb toxicity. sEPO may serve as an early biochemical marker of subclinical Pb toxicity.

Food and nutrient intakes among nomads living in three different areas of Inner Mongolia, China.

The subjects, from Mongolian-style gel tribes, aged 6-79 years, living in three different areas of Inner Mongolia, were asked about their daily food consumption in August 2001 using the 24-hr dietary recall method. There were no significant differences in food intakes between males and females of the same age group in the same area, with a few exceptions, although considerable variation was found among individuals and areas for the amount of foods consumed. Some subjects consumed greater amounts of wheat, vegetables, fruits, and vegetable oils, which suggested they had changed their eating patterns. All subjects drank large quantities of tea, to which was added milk (sheep/goat), cream, butter, parched millet and/or sawa. This traditional tea supplied vitamins and minerals such as vitamin A, iron and calcium.

Food consumption and serum nutritional status of people living in the Kathmandu valley in Nepal.

OBJECTIVE: The food consumption and serum nutritional status of people living in the Kathmandu valley, Nepal, were examined to obtain actual data for comparison with our previous findings. METHODS: A dietary survey of 45 males and 60 females was carried out in March 1997 by the 24-hr dietary recall method and nutrient intake was calculated from food tables of India and Japan. Fasting venous blood samples were obtained and serum biochemical parameters were measured using clinical kits. RESULTS: The mean body mass index values was at about the same level in both sexes, although the mean percentage body fat of females was higher than that of males, and vice versa for packed red cell volume. The main foods consumed by both sexes, were rice, potatoes, meats, milk & dairy products and vegetables, with a difference in the amounts consumed. Females did not drink alcoholic beverages. The mean daily intakes of energy, protein, lipids, iron and vitamin B group for the males were higher than those for the females, while those of vitamins A and C for the males were lower than those for the females. The mean values of serum biochemical parameters for both sexes were generally at the normal levels, but those of ALT and TG were at the higher end of the normal range. Differences of correlation between food groups and between serum parameters were observed depending on the sex, however, no clear relationship between food and nutrient intake and serum biochemical parameters were observed. CONCLUSIONS: The present food intake study revealed that the amounts of food consumption for both sexes, especially for the females, were mostly insufficient although the serum parameters were at the normal levels. The energy intake of both sexes was lower than that of estimated requirements and those in Terai region. The relatively high serum TG level of the subjects may be due to the consumption of large amounts of cereals containing much carbohydrate. Our findings suggested a marked influence on food consumption by food price and income in spite of the easier food availability in the city, and also lack of knowledge about nutrients and health, thus there is need for improvement of the nutritional status of this group of people.

Serum protein and immunoglobulin levels among nepalese living in southern Nepal.

OBJECTIVE: This study examined the levels of serum protein fractionation, immunoglobulin (Ig) and antistreptolysin O (ASO) of people (91 males and 84 females aged 10-68 years) living in the Terai region of southern Nepal, as there has previously been no information available about them. METHODS: Blood samples were collected early in the morning after overnight fasting. Serum protein fractionation was carried out by cellulose-acetate electrophoresis. IgG, IgA, IgM and ASO were measured by immuno-turbidimetry with clinical kits. RESULTS: The mean proportion of albumin (Alb) was rather low due to increased globulin (Glb). The Alb level of males was significantly higher than that of females, while the male γ-Glb level was significantly lower than that of females. The mean values of IgG, IgM and ASO for males were lower than those values for females, although the difference was significant only for the IgM value. The mean values of IgM for female age groups of 30-39 years or less were significantly higher than those for the corresponding male age groups. Age correlated positively with IgA, and negatively with ASO in both sexes. γ-Glb and IgG correlated significantly with TP, AlB, α(1)-Glb and IgA in both sexes. ASO correlated with ß-Glb and IgA in males, and with γ-Glb and IgG in females. CONCLUSIONS: The fact that the level of γ-Glb, a major component of serum globulin, was high suggested exposure to a highly bacterial and viral environment. These results point to the need to prevent infectious diseases as well as improve their nutritional status, especially for children and young adults.

Study on enteric parasitosis and nutritional status of school children in remote hilly areas in Nepal.

We examined the effect of enteric parasitosis on nutritional status of school children in remote hilly areas in Nepal. A total of 325 school children aged 6 to 19 years (boys: 214; girls: 111) who provided both blood and stool samples were included. The height (Ht) (cm), weight (Wt) (kg), age, sex, ethnic of each child were recorded. Fecal samples were examined by direct smear method in duplicate. The blood hemoglobin (Hb) was estimated by Sahli's method in the field (Nepal) whereas the serum protein (Prot), albumin (Alb), Alb/globulin ratio (A/G), serum glutamate pyruvate transaminase (SGPT), cholesterol (Chol), triglycerides (Trig), and serum iron (SI) were measured in Japan. Of the total, 201 (61.8%) had enteric parasites, predominantly the soil transmitted helminths. Ascaris lumbricoides was the most common parasite detected (72.6%) followed by hookworm (16.2%) and others. Protozoan infections were very low (<6.0%). Of the total 201 parasite positive children, 15.4% had multiple infections. No significant differences in Ht and Wt in parasite positive and negative boys and girls of both age groups (< 11 and 11-19 years) were observed (P>0.05). Overall, no significant differences in levels of Hb, Prot, A/G, SGPT, Chol, Trig and SI in the parasite positive and negative children were observed (P>0. 05) but in Alb level (P<0.05). When, the levels of these parameters were compared in the age groups of <11 and 11-19 years, similar findings were observed in both the age groups. But, in the age group of 11-19 years, a significant difference was observed in Trig level (P<0.05). No differences were found in the levels of these parameters in the parasitic positive and negative children belonging to Dalits and different ethnic namely, Indo-Aryan and Tibeto-Burman, respectively.

Study on physical and some serum parameters of inhabitants living in agricultural Terai region, Nepal.

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltranspeptidase (gamma-GTP), lactate dehydrogenase (LDH), urea nitrogen (BUN), uric acid (UA) and creatinine (CRE) levels were examined in 183 people (98 males and 85 females aged 10 to 68 years) living in Terai region in Nepal. The mean values of serum components examined did not differ by sex in the age group of 10-14 years. The mean values of serum AST, ALT and gamma-GTP levels differed significantly between the sexes (P<0.01). The all physical measurements and serum parameters observed correlated well in males, but a few of them correlated in females. The AST-ALT, AST-gamma-GTP, ALT-CRE and BUN-CRE correlated well in both sexes. The UA correlated with ALT and gamma-GTP, CRE with gamma-GTP, LDH and UA in males, while only AST-LDH and gamma-GTP-BUN correlated in females. The levels of most of the serum components examined in this study were within normal ranges for Japanese and others. However, it seemed to be necessary to improve their living conditions as these serum components are related to hepatic or renal function and the infectious diseases.

Serum nutritional status of tocopherol and retinol normalized to lipids of persons living in the southern rural Terai region in Nepal.

OBJECTIVE: The present study examined the levels of serum α-Tocopherol (Toc), retinol (Ret), cholesterol (Chol) and triglycerides (TG), and their correlations in the sera of people in Nepal. METHODS: The survey was conducted on the general populace in the agricultural Terai region in southern Nepal. The study population consisted of 93 males and 83 females aged 10-68 years. Serum Toc and Ret were measured by high-performance liquid chromatography. RESULTS: No significant differences were observed between the genders for the average of total Chol (T-Chol) (140 and 145 mg/100 ml, respectively), HDL-C (45 and 47 mg/100 ml), LDL-C (94 and 97 mg/100 ml), and TG (106 and 110 mg/100 ml), and the ratio of LDL/HDL (2.16). The levels of mean Toc (4.32 and 4.27 µg/ml) were about the same for both genders, while the mean Ret levels were significantly higher for males (624 ng/ml) than for females (535 ng/ml) (p<0.001). A direct relationship was found between the levels of Toc and Ret (r=0.46, p<0.001 and r=0.28, p<0.05 for males and females, respectively). Serum levels of Toc and Ret were positively related to the levels of Chol (r=0.48 and r=0.58, p<0.001 for males and r=0.49, p<0.01 and r-0.28, p<0.05 for females, respectively). The ratio of Toc/TG normalized to serum TG was directly correlated to the ratio of Ret/TG (r=0.79 for males, and r=0.72 for females, p<0.001, respectively) and the ratios of Toc/TG and Ret/TG were negatively related to the LDL/HDL levels (r=-0.49 and r=-0.43, for males, and r=-0.46 and r=-0.57 for females, p<0.001, respectively). CONCLUSION: The levels of Toc and Ret were low in the sera of people living in the southern rural Terai region in Nepal, and it was found that lower levels of Toc and Ret normalized to TG increased the ratio of LDL/HDL. These results suggest that greater intake of foods rich in Toc and Ret should be encouraged to reduce the erisk of coronary heart disease.