Proponents of the recognition concept of species hold that isolating mechanisms, including chromosome rearrangements, play no role in speciation while the more commonly accepted biological species concept proposes that isolation mechanisms are instrumental in the formation of new species. Moreover, some adherents of the biological concept of species, reject the hypothesis that chromosomal rearrangements can be instrumental in causing reproductive isolation and, hence, speciation. Evidence to the causative role played in speciation by chromosome changes can be obtained from cytogenetic investigations of sibling species, in parallel with analyses of gene products, DNA polymorphism and premating behaviour. This study reports the results of a cytogenetic investigation of 97 specimens of the vlei rat 0. irroratus, from 18 South African localities, and 11 samples of the Angoni vlei rat 0. angoniensis from two geographically distant populations. All 0. angoniensis individuals showed a constant karyotype with 56 acrocentric chromosomes but extensive variation was detected in 0. irroratus. Five cytotypes could be recognized within the latter. In the south-eastern parts of its South African range, 0. irroratus had a diploid number (2n) of 30 chromosomes in whicll all autosomes were acrocentric (cytotype A) while further east (cytotype A2), the diploid number was 30-32 with, again, acrocentric autosomes, A further acrocentric cytotype (AI) with 2n = 24-27 occupied the southern and south-eastern slopes of the Drakensberg range. A type with 2n = 28-30 (cytotype B), with eight pairs of biarmed autosomes, was found in the southern Cape region while in the Cape of Good Hope and in the north-eastern parts of South Africa, 0. irroratus had 2n = 28 with only four pairs of biarmed autosomes (cytotype C). Most of the numerical changes were due to variation in the number of copies of Bchromosomes which were small, biarmed and partly heterochromatic. C-banding analysis revealed that the short arms of bianned autosomes were totally heterochromatic. On the other hand, G-banding patterns of acrocentric autosomes were, with two exceptions (AI and A2 types), similar in all cytotypes while G~banding of the long arms VII of biarmed chromosomes matched the pattern of their homologues in acrocentric cytotypes. A potentially heterotic rearrangement was detected in the Al localities where a unique acrocentric autosome was identified as the product of a fusion between chromosomes 7 and 12. The geographic distribution of these groups of karyotypes correlated, by Discriminant Function Analysis, with bioclimatic regions of South Africa. The Al cytotype was shown to occupy the coldest and wettest region of the montane Drakensberg while the B type is found in the hot area of the eastern Cape with an unpredictable rainfall pattern: group C occupies regions of intermediate climate. Gene product analysis was carried out using the novel approach of subjecting liver homogenates to "Western blotting". This method was first assessed at supraspecilic level using specimens of various southern African rodents, and allowed the generation of phylogenies essentially similar to those produced by allozyme studies of the same taxa. At intraspecilic level, immunobloHing analysis did not reveal synapomorphies congruent with karyotype groups. This was interpreted, in conjunction with available allozyme data from the same populations, as evidence of low genetic differentiation between 0. irroratus cytotypes, A measure of genetic divergence was indicated in two populations from the Cape province and this was in agreement with existing data from allozyme electrophoresis and mitochondrial DNA polymorphism. The cytogenetic results were related to available data on breeding and premating behaviour concerning some of the O. irroratus populations investigated here. The presence of the 7/12 chromosome fusion in the Al cytotype correlated with a dramatic reproductive impairment of FI individuals originated from Al/A2 and Al/B cytotype crosses. Evidence of partial premating behavioural barriers has been reported by others, but information on premating behaviour between populations which are not chromosomally isolated is lacking. Therefore, it was not possible to establish if behavioural premating barriers preceded, or followed, the fixation of negatively heterotic chromosomal rearrangements. It was, nevertheless, suggested that the existence of such impaired mate recognition may be an example of reproductive character displacement which may have followed the fixation of the t(7: 12) typiVIII cal of the Al populations. In conclusion, the existence of chromosome changes in the AI, and possibly A2, populations accompanied by low genetic divergence and severely impaired hybrid reproductive success, are consistent with a hypothesis whereby chromosomal reproductive isolation causes speciation. Nonetheless, other speciation mechanisms mediated by genetic divergence and/or mate recognition failure, are possible in other populations where no chromosome changes of negatively heterotic potential were found. / Thesis (Ph.D.)-University of Natal, 1996.

The imbalance in the levels of certain microRNAs (miRNAs) in Alzheimer’s disease (AD) brains promotes alterations in tau proteostasis and neurodegeneration. However, potential mechanisms governing how specific miRNAs are dysregulated in AD brains are still under investigation. Epitranscriptomics is a mode of post-transcriptional regulation that can control brain functions during development and adulthood. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases that has the ability to modify mammalian non-coding RNAs. Importantly, autosomal-recessive loss of function mutations in NSun2 have been associated with neurological abnormalities in humans. Here, we report that dysregulation of NSun2 can induce alterations in tau phosphorylation by modulating the levels of miR-125b, a main player in tau pathology. We were able to provide supporting evidence by utilizing several model systems such as Drosophila, human induced pluripotent stem cell (iPSC) derived neurons, rat primary neuronal cultures and mice. Our Western blot analysis not only shows that NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex, but also NSun2 protein expression levels are downregulated in post-mortem brain tissues from AD patients. Remarkably, we also found decreased NSun2 protein levels in AD mice and human cellular models. To prove these observed alterations were unique to AD, we further evaluated brain tissues from other tauopathies. Strikingly, NSun2 protein levels were similar between tauopathy cases and controls indicating that dysregulation of NSun2 might be unique to AD cases. Further, we investigated the pathological role of NSun2 by utilizing a well-established Drosophila melanogaster model of tau-induced toxicity. We found that a reduction of NSun2 protein levels exacerbated tau toxicity while overexpression of NSun2 partially abrogated toxicity proving bidirectionality. We used a lentiviral system to knock down NSun2 expression in iPSC derived neuronal cultures. Western blot analysis and immunofluorescence staining showed a significant change in tau phosphorylation levels. To investigate what could be triggering observed alterations in NSun2 levels, we performed experiments in rat primary hippocampal neurons. We found that the treatment with oligomeric amyloid-beta A caused a decrease in NSun2 protein levels and at the same time, increased tau phosphorylation levels in primary hippocampal neurons. Lastly, we performed RNA immunoprecipitation coupled with qPCR and histological analysis using NSun2 conditional knockout (KO) mice and observed that NSun2 deficiency promoted aberrant levels of m6A methylated miR-125b and tau hyperphosphorylation. Altogether, our study demonstrates that neuronal NSun2 deficiency in AD promotes neurodegeneration by altering tau phosphorylation and tau toxicity through an epitranscriptomic regulatory mechanism and highlights a potential novel therapeutic target.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple lines of High Alcohol Preferring (HAP) mice were selectively bred for their intake of 10% ethanol (v/v) during 24-h daily access over a four-week period, with the highest drinking lines exhibiting intakes in excess of 20 g/kg/day. Drinking rhythms and corresponding blood ethanol concentrations (BEC) of the highest drinking HAP lines to those of the C57BL/6J (B6) inbred strain. Adult male and female crossed HAP (cHAP), HAP1 and B6 mice had free-choice access to 10% ethanol and water for 3 weeks prior to bi-hourly assessments of intake throughout the dark portion of a reverse 12:12 light dark cycle. In another cohort of cHAP mice, the same procedure was used to assess bi-hourly ethanol intake, and blood samples were taken across the day to look at the pattern of accumulation in these mice. Finally, considering the high level of intake by cHAP mice, we were interested in assessing whether metabolic and functional tolerance develop following chronic free-choice access, which were assessed using 2.0 and 1.75 g/kg challenge doses of 20% ethanol, respectively. cHAP and HAP1 mice maintained an excessive level of intake throughout the dark portion of the cycle, accumulating mean BEC levels of 261.5 + 18.09 and 217.9 + 25.02 mg/dl at 7-8 hours following lights off, respectively. B6 mice drank comparatively modestly, and did not accumulate high BEC levels (53.63 + 8.15 mg/dl). In the cHAP cohort, mean BECs were 112.47 + 19.91 at 2 hours after lights off, 189.00 + 27.40 at 6 hours after lights off, 193.80 + 29.66 at 10 hours after lights off, and 89.68 + 22.19 at 2 hours after lights on. Further, following 3 weeks of ethanol access, cHAP mice had a faster rate of ethanol metabolism and fewer hind slips than water-only exposed mice (ps < .05). In conclusion, the excessive free-choice drinking demonstrated by the HAP1 and cHAP lines, as well as the pattern of sustained high BECs in cHAP mice, challenge the notion that rodents will not reliably and voluntarily sustain ethanol intake at pharmacologically relevant levels. These results suggest that the highest drinking HAP lines may provide a unique opportunity for modeling the excessive intake that has been observed in alcohol-dependent individuals. Further, we observed that cHAP mice develop both metabolic and functional tolerance to the ataxic effects of ethanol following 3 weeks of free-choice access. Together, these findings support HAP mice as translational rodent model of alcoholism, and provide rationale for exploration of the predisposing factors for excessive consumption, as well as the development of physiological, behavioral, and toxicological outcomes following alcohol exposure.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcoholism inheritance rates have been estimated as high as 60% in a human population. Many significant features of alcohol dependence have been replicated in rodent animal models of alcoholism, however not in totality. These animal models include inbred preferring (iP) and nonpreferring (iNP) rat types. Congenic rats have been engineered from the iP and iNP strains whereby a P congenic rat has in its genome a well-chosen chromosomal portion taken from an NP rat (P.NP) and, reciprocally, an NP congenic rat has acquired the analogous DNA from a P rat (NP.P). In this case, a quantitative trait locus (QTL) from chromosome 4 is the donor genetic material for the congenic rats. It is of great interest to further study this chromosome 4 QTL because it has been found to control a significant portion of ethanol consumption behavior in iP and iNP rats. This study aimed to behaviorally profile the iP, iNP and reciprocal congenic rats. As a result of the behavioral profiling of these genetically related groups, some conclusions could be made regarding which behaviors appear to be controlled by the chromosome 4 donor DNA.This study primarily utilized the Multivariate Concentric Square Field apparatus (MCSF) to characterize behavioral profiles for the inbred and congenic rats. The Open field (OF) and Elevated plus maze (EPM) supported this effort. The MCSF is valuable in that it allows for the animals to interact within an environment that has ethological value. The 12 different zones that make up the field are characterized by some functional quality in terms of type and duration of behavior performed, etc. The behavioral data is aggregated and finally represented in terms of five functional categories, the elements of the behavioral profile: general activity, exploratory activity, risk assessment, risk taking, and shelter seeking. The study hypotheses were shaped by prior research suggesting that iPs should display lower general activity and risk taking strategy than iNPs in the MCSF. Inbred Ps should be more active in the OF and spend more time in the center of the EPM. Generally, it is expected that the iP QTL confer behavioral phenotypes to the iNP strain that deviate toward a "P" behavioral phenotype and reciprocally, the iNP QTL confer behavioral phenotypes to the iP strain that deviate toward an "NP" behavioral phenotype. The results showed that iP rats performed more risk assessment and risk taking behavior and less shelter seeking and anxiety-like behavior than iNP rats. It followed that P.NP congenic rats significantly downgraded their risk assessment and risk taking behavior when compared to iP rats. This decrease can be attributed to the chromosome 4 QTL donated from the iNP breed. All together this study concludes that risk assessment and risk taking behavior in the iP rats is controlled by the same DNA region that, in part, determines voluntary intake of ethanol consumption. Further fine mapping of the QTL region should help in discovering if the same DNA sequences that influence ethanol intake also significantly influence risk behavior.