Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior.

Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1+) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1+ mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1+ mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (-/-) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1+ mice, the Atp8a1 (-/-) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.

A novel p.Leu(381)Phe mutation in presenilin 1 is associated with very early onset and unusually fast progressing dementia as well as lysosomal inclusions typically seen in Kufs disease.

Whole exome sequencing in a family with suspected dominant Kufs disease identified a novel Presenilin 1 mutation p.Leu(381)Phe in three brothers who, along with their father, developed progressive dementia and motor deficits in their early 30 s. All affected relatives had unusually rapid disease progression (on average 3.6 years from disease onset to death). In silico analysis of mutation p.Leu(381)Phe predicted more detrimental effects when compared to the common Presenilin 1 mutation p.Glu(280)Ala. Electron microscopy study of peripheral fibroblast cells of the proband showed lysosomal inclusions typical for Kufs disease. However, brain autopsy demonstrated typical changes of Alzheimer's disease.

Mutations in the gene DNAJC5 cause autosomal dominant Kufs disease in a proportion of cases: study of the Parry family and 8 other families.

BACKGROUND: The Neuronal Ceroid Lipofuscinoses (NCL) comprise at least nine progressive neurodegenerative genetic disorders. Kufs disease, an adult-onset form of NCL may be recessively or dominantly inherited. Our study aimed to identify genetic mutations associated with autosomal dominant Kufs disease (ADKD). METHODOLOGY AND PRINCIPAL FINDINGS: We have studied the family first reported with this phenotype in the 1970s, the Parry family. The proband had progressive psychiatric manifestations, seizures and cognitive decline starting in her mid 20 s. Similarly affected relatives were observed in seven generations. Several of the affected individuals had post-mortem neuropathological brain study confirmatory for NCL disease. We conducted whole exome sequencing of three affected family members and identified a pLeu116del mutation in the gene DNAJC5, which segregated with the disease phenotype. An additional eight unrelated affected individuals with documented autosomal dominant or sporadic inheritance were studied. All had diagnostic confirmation with neuropathological studies of brain tissue. Among them we identified an additional individual with a p.Leu115Arg mutation in DNAJC5. In addition, a pAsn477Ser change in the neighboring gene PRPF6, a gene previously found to be associated with retinitis pigmentosa, segregated with the ADKD phenotype. Interestingly, two individuals of the Parry family did report visual impairment. CONCLUSIONS: Our study confirmed the recently reported association of DNAJC5 mutations with ADKD in two out of nine well-defined families. Sequence changes in PRPF6 have not been identified in other unrelated cases. The association of vision impairment with the expected PRPF6 dysfunction remains possible but would need further clinical studies in order to confirm the co-segregation of the visual impairment with this sequence change.

Digallate dimers of (-)-epigallocatechin gallate inactivate herpes simplex virus.

Topical microbicides are potentially an alternative method to vaccines for reducing the spread of herpes simplex virus (HSV). We have previously shown (S. Liu et al., Biochim. Biophys. Acta 1723:270-281, 2005) that the catechin (-)-epigallocatechin gallate (EGCG) inactivates HSV at neutral pH; however, to function in the female genital tract EGCG must also be effective at acidic pH. EGCG inactivated HSV-1 and HSV-2 at pH 8.0 by 3 log(10) to 4 log(10) but was ineffective at pH 5.7. The EGCG digallate dimers theasinensin A, P2, and theaflavin-3,3'-digallate (TF-3) inactivated both viruses by 3 log(10) to 4 log(10) at pH 5.7 and as much as 5 log(10) at pH 8.0. TF-3 inactivated HSV-1 and HSV-2 by 4 to 5 log(10) in the pH range of 4.0 to 5.7. Dimers with one gallate moiety had antiviral activity intermediate between the activities of EGCG and digallate dimers. Confocal and electron microscopy showed that theasinensin A did not damage Vero cells. All EGCG dimers inactivated enveloped viruses with class I, class II, and class III (HSV-1, HSV-2) fusion proteins more effectively than did monomeric EGCG. EGCG had no activity against the nonenveloped viruses tested, but TF-3 reduced the titer of 4 of 5 nonenveloped viruses by ≅2 to 3.5 log(10). Results also showed that HSV-1 glycoprotein B (gB) was aggregated more rapidly by theasinensin A than EGCG, which, when taken together with the nonenveloped virus data, suggests that dimers may inhibit the function of viral proteins required for infectivity. Digallate dimers of EGCG appear to have excellent potential as microbicidal agents against HSV at acidic and neutral pHs.

Chlorination byproducts induce gender specific autistic-like behaviors in CD-1 mice.

In 2000, the Agency for Toxic Substances and Disease Registry (ATSDR) released a report concerning elevated autism prevalence and the presence water chlorination byproducts in the municipal drinking water supply in Brick Township, New Jersey. The ATSDR concluded that it was unlikely that these chemicals, specifically chloroform, bromoform (Trihalomethanes; THMs) and tetrachloroethylene (Perchloroethylene; PCE) had contributed to the prevalence of autism in this community based upon correlations between timing of exposure and/or concentration of exposure. The ATSDR conclusion may have been premature, as there is no conclusive data evidencing a correlation between a particular developmental time point that would render an individual most susceptible to toxicological insult with the development of autism. Therefore, it was our aim to determine if these chemicals could contribute to autistic like behaviors. We found that males treated with THMs and PCE have a significant reduction in the number of ultrasonic vocalizations (USVs) emitted in response to maternal separation, which are not attributed to deficits in vocal ability to or to lesser maternal care. These same males also show significantly elevated anxiety, an increase in perseverance behavior and a significant reduction in sociability. The sum of our data suggests that male, but not female mice, develop autistic like behaviors after gestational and postnatal exposure to the aforementioned chemical triad via drinking water. We believe development of such aberrant behaviors likely involves GABAergic system development.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice.

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

Epigallocatechin gallate inactivates clinical isolates of herpes simplex virus.

In the absence of a fully effective herpes simplex virus (HSV) vaccine, topical microbicides represent an important strategy for preventing HSV transmission. (-)-Epigallocatechin gallate (EGCG) (molecular weight, 458.4) is the primary catechin in green tea. The present study shows that EGCG has greater anti-HSV activity than other green tea catechins and inactivates multiple clinical isolates of HSV type 1 (HSV-1) and HSV-2. EGCG reduced HSV-2 titers by >or=1,000-fold in 10 to 20 min and reduced HSV-1 titers by the same amount in 30 to 40 min. The anti-HSV activity of EGCG is due to a direct effect on the virion, and incubating Vero and CV1 cells with EGCG for 48 h prior to infection with HSV-1 and HSV-2, respectively, does not reduce HSV production. Electron microscopic (EM) studies showed that purified virions exposed to EGCG were damaged, and EM immunogold labeling of the envelope glycoproteins gB and gD was significantly reduced following EGCG treatment while capsid protein labeling was unchanged. When purified HSV-1 envelope glycoproteins gB and gD were incubated with EGCG and then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lower-molecular-weight gB and gD bands decreased and new higher-molecular-weight bands appeared, indicating the EGCG-dependent production of macromolecular complexes. gB and gD are essential for HSV infectivity, and these results suggest that EGCG could inactivate HSV virions by binding to gB, gD, or another envelope glycoprotein. EGCG is stable in the pH range found in the vagina and appears to be a promising candidate for use in a microbicide to reduce HSV transmission.

Scanning electron microscopy of the infundibulum, ampulla, and eggs of mice.

The objective of the study reported here was to use scanning electron microscopy (SEM) to discover and describe the details of three-dimensional profiles and the natural (not surgically disturbed) topography/location of the infundibulum in the mouse. It will help new investigators to more quickly identify the infundibulum for successful transfer of microinjected eggs through a small opening into the oviduct/ampulla of pseudopregnant female mice for producing transgenic mice. Results of the study also illustrate the geographic orientation and natural topographic features of the ovary, infundibulum, ampulla, oviduct, and uterus. The presence of cilia on the surface of the crown foldings in the longitudinal section of the infundibular head stained with 1% toluidine blue provided direct evidence that evagination of the internal cilia of the infundibulum/oviduct results in formation of the infundibular crown. The new observation of the narrowing region of the infundibular head after surgical removal of the crown also suggests that formation of the infundibular crown may have resulted from the "evagination process" of internal cilia of the infundibulum/oviduct surface. The results also provide new evidence that the crown, terminal opening, and appearance of the left and the right infundibula of the same mouse differ.

Brush-like fibers on the human chromosome periphery.

A brush-like border apparently composed of fibers protruding from metaphase chromosomes of human lymphocytes was observed for the first time using transmission (TEM) and scanning electron microscopy (SEM). On the basis of size and sensitivity to colcemid, the fibers may be related to microtubules and spindle organization. The brush-like fibers were observed when chemically fixed metaphase chromosome spreads were placed on glass slides and maintained under "wet" conditions (not allowed to air dry after fixation for conventional cytogenetic protocols) until postfixation protocols for TEM and SEM were performed. The purpose of this study was to establish the occurrence of the brush-like fibers and to determine the effects of colcemid on these fibers.