Impairment of DHA synthesis alters the expression of neuronal plasticity markers and the brain inflammatory status in mice.

Docosahexaenoic acid (DHA) is a ω-3 fatty acid typically obtained from the diet or endogenously synthesized through the action of elongases (ELOVLs) and desaturases. DHA is a key central nervous system constituent and the precursor of several molecules that regulate the resolution of inflammation. In the present study, we questioned whether the impaired synthesis of DHA affected neural plasticity and inflammatory status in the adult brain. To address this question, we investigated neural and inflammatory markers from mice deficient for ELOVL2 (Elovl2-/- ), the key enzyme in DHA synthesis. From our findings, Elovl2-/- mice showed an altered expression of markers involved in synaptic plasticity, learning, and memory formation such as Egr-1, Arc1, and BDNF specifically in the cerebral cortex, impacting behavioral functions only marginally. In parallel, we also found that DHA-deficient mice were characterized by an increased expression of pro-inflammatory molecules, namely TNF, IL-1ß, iNOS, caspase-1 as well as the activation and morphologic changes of microglia in the absence of any brain injury or disease. Reintroducing DHA in the diet of Elovl2-/- mice reversed such alterations in brain plasticity and inflammation. Hence, impairment of systemic DHA synthesis can modify the brain inflammatory and neural plasticity status, supporting the view that DHA is an essential fatty acid with an important role in keeping inflammation within its physiologic boundary and in shaping neuronal functions in the central nervous system.

Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior.

Hutchinson-Gilford progeria syndrome (HGPS) is a segmental progeroid syndrome with multiple features suggestive of premature accelerated aging. Accumulation of progerin is thought to underlie the pathophysiology of HGPS. However, despite ubiquitous expression of lamin A in all differentiated cells, the HGPS mutation results in organ-specific defects. For example, bone and skin are strongly affected by HGPS, while the brain appears to be unaffected. There are no definite explanations as to the variable sensitivity to progeria disease among different organs. In addition, low levels of progerin have also been found in several tissues from normal individuals, but it is not clear if low levels of progerin contribute to the aging of the brain. In an attempt to clarify the origin of this phenomenon, we have developed an inducible transgenic mouse model with expression of the most common HGPS mutation in brain, skin, bone and heart to investigate how the mutation affects these organs. Ultrastructural analysis of neuronal nuclei after 70 weeks of expression of the LMNA c.1824C>T mutation showed severe distortion with multiple lobulations and irregular extensions. Despite severe distortions in the nuclei of hippocampal neurons of HGPS animals, there were only negligible changes in gene expression after 63 weeks of transgenic expression. Behavioral analysis and neurogenesis assays, following long-term expression of the HGPS mutation, did not reveal significant pathology. Our results suggest that certain tissues are protected from functional deleterious effects of progerin.

High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio.

At present, there are few means to track symptomatic stages of CNS aging. Thus, although metabolic changes are implicated in mtDNA mutation-driven aging, the manifestations remain unclear. Here, we used normally aging and prematurely aging mtDNA mutator mice to establish a molecular link between mitochondrial dysfunction and abnormal metabolism in the aging process. Using proton magnetic resonance spectroscopy and HPLC, we found that brain lactate levels were increased twofold in both normally and prematurely aging mice during aging. To correlate the striking increase in lactate with tissue pathology, we investigated the respiratory chain enzymes and detected mitochondrial failure in key brain areas from both normally and prematurely aging mice. We used in situ hybridization to show that increased brain lactate levels were caused by a shift in transcriptional activities of the lactate dehydrogenases to promote pyruvate to lactate conversion. Separation of the five tetrameric lactate dehydrogenase (LDH) isoenzymes revealed an increase of those dominated by the Ldh-A product and a decrease of those rich in the Ldh-B product, which, in turn, increases pyruvate to lactate conversion. Spectrophotometric assays measuring LDH activity from the pyruvate and lactate sides of the reaction showed a higher pyruvate → lactate activity in the brain. We argue for the use of lactate proton magnetic resonance spectroscopy as a noninvasive strategy for monitoring this hallmark of the aging process. The mtDNA mutator mouse allows us to conclude that the increased LDH-A/LDH-B ratio causes high brain lactate levels, which, in turn, are predictive of aging phenotypes.

Bouts of rest and physical activity in C57BL/6J mice.

The objective was to exploit the raw data output from a scalable home cage (type IIL IVC) monitoring (HCM) system (DVC®), to characterize pattern of undisrupted rest and physical activity (PA) of C57BL/6J mice. The system's tracking algorithm show that mice in isolation spend 67% of the time in bouts of long rest (≥40s). Sixteen percent is physical activity (PA), split between local movements (6%) and locomotion (10%). Decomposition revealed that a day contains ˜7100 discrete bouts of short and long rest, local and locomotor movements. Mice travel ˜330m per day, mainly during the dark hours, while travelling speed is similar through the light-dark cycle. Locomotor bouts are usually <0.2m and <1% are >1m. Tracking revealed also fits of abnormal behaviour. The starting positions of the bouts showed no preference for the rear over the front of the cage floor, while there was a strong bias for the peripheral (75%) over the central floor area. The composition of bouts has a characteristic circadian pattern, however, intrusive husbandry routines increased bout fragmentation by ˜40%. Extracting electrode activations density (EAD) from the raw data yielded results close to those obtained with the tracking algorithm, with 81% of time in rest (<1 EAD s-1) and 19% in PA. Periods ≥40 s of file when no movement occurs and there is no EAD may correspond to periods of sleep (˜59% of file time). We confirm that EAD correlates closely with movement distance (rs>0.95) and the data agreed in ˜97% of the file time. Thus, albeit EAD being less informative it may serve as a proxy for PA and rest, enabling monitoring group housed mice. The data show that increasing density from one female to two males, and further to three male or female mice had the same effect size on EAD (˜2). In contrast, the EAD deviated significantly from this stepwise increase with 4 mice per cage, suggesting a crowdedness stress inducing sex specific adaptations. We conclude that informative metrics on rest and PA can be automatically extracted from the raw data flow in near-real time (< 1 hrs). As discussed, these metrics relay useful longitudinal information to those that use or care for the animals.

Major oscillations in spontaneous home-cage activity in C57BL/6 mice housed under constant conditions.

The mouse is the most important mammalian model in life science research and the behavior of the mouse is a key read-out of experimental interventions and genetic manipulations. To serve this purpose a solid understanding of the mouse normal behavior is a prerequisite. Using 14-19 months of cumulative 24/7 home-cage activity recorded with a non-intrusive technique, evidence is here provided for a highly significant circannual oscillation in spontaneous activity (1-2 SD of the mean, on average 65% higher during peak of highs than lows; P = 7E-50) of male and female C57BL/6 mice held under constant conditions. The periodicity of this hitherto not recognized oscillation is in the range of 2-4 months (average estimate was 97 days across cohorts of cages). It off-sets responses to environmental stimuli and co-varies with the feeding behavior but does not significantly alter the preference for being active during the dark hours. The absence of coordination of this rhythmicity between cages with mice or seasons of the year suggest that the oscillation of physical activity is generated by a free-running intrinsic oscillator devoid of external timer. Due to the magnitude of this rhythmic variation it may be a serious confounder in experiments on mice if left unrecognized.

Multipotent mesenchymal stromal cells attenuate chronic inflammation and injury-induced sensitivity to mechanical stimuli in experimental spinal cord injury.

PURPOSE: Previous reports established that after a contusion injury to the rat spinal cord, locomotor function was enhanced by the transplantation of cells from bone marrow referred to as either mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). It has also been established that neural stem cells (NSCs) enhance locomotor function after transplantation into the injured rat spinal cord. However, the beneficial effects of NSCs are limited by graft-induced allodynia-like responses. Little is known about the effects of MSCs on sensory function in spinal cord injury. Therefore, the objective of this research was to determine whether transplantation of MSCs into the injured rat spinal cord induces allodynia-like responses. METHODS: Contusion injuries of two different severities were induced in rats to examine the effects of transplantation with MSCs on sensorimotor deficits. The effects of MSCs on chronic inflammation were investigated, since inflammation is reported to have a role in the sensorimotor deficits associated with spinal cord injury. In addition, observations in other models suggest that MSCs possess immunosuppressive effects. RESULTS: We found that in contrast to previous observations with the transplantation of neural stem cells, transplantation of MSCs did not induce allodynia. MSCs attenuated injury-induced sensitivity to mechanical stimuli but had no effect on injury-induced sensitivity to cold stimuli. MSCs also significantly attenuated the chronic inflammatory response as assayed by GFAP immunoreactivity for reactive astrocytes and ED1 immunoreactivity for activated macrophages/microglia. In addition, transplantation of MSCs increased white matter volumes and decreased cyst size in sections of the cord containing the lesion. CONCLUSION: The results suggest that the sensorimotor enhancements produced by MSCs can at least in part be explained by anti-inflammatory/immunosuppressive effects of the cells, similar to such effects of these cells observed in other experimental models.

Lrrk2 and alpha-synuclein are co-regulated in rodent striatum.

LRRK2, alpha-synuclein, UCH-L1 and DJ-1 are implicated in the etiology of Parkinson's disease. We show for the first time that increase in striatal alpha-synuclein levels induce increased Lrrk2 mRNA levels while Dj-1 and Uch-L1 are unchanged. We also demonstrate that a mouse strain lacking the dopamine signaling molecule DARPP-32 has significantly reduced levels of both Lrrk2 and alpha-synuclein, while mice carrying a disabling mutation of the DARPP-32 phosphorylation site T34A or lack alpha-synuclein do not show any changes. To test if striatal dopamine depletion influences Lrrk2 or alpha-synuclein expression, we used the neurotoxin 6-hydroxydopamine in rats and MitoPark mice in which there is progressive degeneration of dopamine neurons. Because striatal Lrrk2 and alpha-synuclein levels were not changed by dopamine depletion, we conclude that Lrrk2 and alpha-synuclein mRNA levels are possibly co-regulated, but they are not influenced by striatal dopamine levels.

Towards large scale automated cage monitoring - Diurnal rhythm and impact of interventions on in-cage activity of C57BL/6J mice recorded 24/7 with a non-disrupting capacitive-based technique.

BACKGROUND AND AIMS: Automated recording of laboratory animal's home cage behavior is receiving increasing attention since such non-intruding surveillance will aid in the unbiased understanding of animal cage behavior potentially improving animal experimental reproducibility. MATERIAL AND METHODS: Here we investigate activity of group held female C57BL/6J mice (mus musculus) housed in standard Individually Ventilated Cages across three test-sites: Consiglio Nazionale delle Ricerche (CNR, Rome, Italy), The Jackson Laboratory (JAX, Bar Harbor, USA) and Karolinska Insititutet (KI, Stockholm, Sweden). Additionally, comparison of female and male C57BL/6J mice was done at KI. Activity was recorded using a capacitive-based sensor placed non-intrusively on the cage rack under the home cage collecting activity data every 250 msec, 24/7. The data collection was analyzed using non-parametric analysis of variance for longitudinal data comparing sites, weekdays and sex. RESULTS: The system detected an increase in activity preceding and peaking around lights-on followed by a decrease to a rest pattern. At lights off, activity increased substantially displaying a distinct temporal variation across this period. We also documented impact on mouse activity that standard animal handling procedures have, e.g. cage-changes, and show that such procedures are stressors impacting in-cage activity. These key observations replicated across the three test-sites, however, it is also clear that, apparently minor local environmental differences generate significant behavioral variances between the sites and within sites across weeks. Comparison of gender revealed differences in activity in the response to cage-change lasting for days in male but not female mice; and apparently also impacting the response to other events such as lights-on in males. Females but not males showed a larger tendency for week-to-week variance in activity possibly reflecting estrous cycling. CONCLUSIONS: These data demonstrate that home cage monitoring is scalable and run in real time, providing complementary information for animal welfare measures, experimental design and phenotype characterization.

Inhibiting epidermal growth factor receptor improves structural, locomotor, sensory, and bladder recovery from experimental spinal cord injury.

Lack of axon regeneration in the adult CNS has been attributed partly to myelin inhibitors and the properties of astrocytes. After spinal cord injury, proliferating astrocytes not only represent a physical barrier to regenerating axons but also express and secrete molecules that inhibit nerve growth, including chondroitin sulfate proteoglycans (CSPGs). Epidermal growth factor receptor (EGFR) activation triggers astrocytes into becoming reactive astrocytes, and EGFR ligands stimulate the secretion of CSPGs as well as the formation of cribriform astrocyte arrangements that contribute to the formation of glial scars. Recently, it was shown that EGFR inhibitors promote nerve regeneration in vitro and in vivo. Blocking a novel Nogo receptor interacting mechanism and/or effects of EGFR inhibition on astrocytes may underlie these effects. Here we show that rats subjected to weight-drop spinal cord injury can be effectively treated by direct delivery of a potent EGFR inhibitor to the injured area, leading to significantly better functional and structural outcome. Motor and sensory functions are improved and bladder function is restored. The robust effects and the fact that other EGFR inhibitors are in clinical use in cancer treatments make these drugs particularly attractive candidates for clinical trials in spinal cord injury.

Recovery from spinal cord injury in tumor necrosis factor-alpha, signal transducers and activators of transcription 4 and signal transducers and activators of transcription 6 null mice.

Tumor necrosis factor-alpha is a central cytokine involved in the regulation of the innate immune response. Signal transducers and activators of transcription 4 and signal transducers and activators of transcription 6 are second messengers mediating the Th1 and Th2-specific immune responses, respectively. We studied the outcome of spinal cord injury with respect to the locomotion and axonal regeneration in tumor necrosis factor-alpha, signal transducers and activators of transcription 4 and signal transducers and activators of transcription 6 knockout mice. Locomotor behavior after injury differed between mouse strains, but not between wild-type and the knockout genotypes of the same strain. Regeneration of descending tracts, assessed by fluorogold/fluororuby retrograde double-labeling, however, appeared hampered by Th2 deficiency.

Association study of two genetic variants in mitochondrial transcription factor A (TFAM) in Alzheimer's and Parkinson's disease.

Mitochondrial (mt) dysfunction has been implicated in Alzheimer's (AD) and Parkinson's disease (PD). Mitochondrial transcription factor A (TFAM) is needed for mtDNA maintenance, regulating mtDNA copy number and is absolutely required for transcriptional initiation at mtDNA promoters. Two genetic variants in TFAM have been reported to be associated with AD in a Caucasian case-control material collected from Germany, Switzerland and Italy. One of these variants was reported to show a tendency for association with AD in a pooled Scottish and Swedish case-control material and the other variant was reported to be associated with AD in a recent meta-analysis. We investigated these two genetic variants, rs1937 and rs2306604, in an AD and a PD case-control material, both from Sweden and found significant genotypic as well as allelic association to marker rs2306604 in the AD case-control material (P=0.05 and P=0.03, respectively), where the A-allele appears to increase risk for developing AD. No association was observed for marker rs1937. We did not find any association in the PD case-control material for either of the two markers. The distribution of the two-locus haplotype frequencies (based on rs1937 and rs2306604) did not differ significantly between affected individuals and controls in the two sample sets. However, the global P-value for haplotypic association testing indicated borderline association in the AD sample set. Our data suggests that the rs2306604 A-allele could be a moderate risk factor for AD, which is supported by the recent meta-analysis.

Glial cell line-derived neurotrophic factor partially ameliorates motor symptoms without slowing neurodegeneration in mice with respiratory chain-deficient dopamine neurons.

Degeneration of midbrain dopamine neurons causes the striatal dopamine deficiency responsible for the hallmark motor symptoms of Parkinson's disease (PD). Intraparenchymal delivery of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), is a possible future therapeutic approach. In animal PD models, GDNF can both ameliorate neurodegeneration and promote recovery of the dopamine system following a toxic insult. However, clinical studies have generated mixed results, and GDNF has not been efficacious in genetic animal models based on α-synuclein overexpression. We have tested the response to GDNF in a genetic mouse PD model with progressive degeneration of dopamine neurons caused by mitochondrial impairment. We find that GDNF, delivered to the striatum by either an adeno-associated virus or via miniosmotic pumps, partially alleviates the progressive motor symptoms without modifying the rate of neurodegeneration. These behavioral changes are accompanied by increased levels of dopamine in the midbrain, but not in striatum. At high levels, GDNF may instead reduce striatal dopamine levels. These results demonstrate the therapeutic potential of GDNF in a progressively impaired dopamine system.

Adh1 and Adh1/4 knockout mice as possible rodent models for presymptomatic Parkinson's disease.

Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinson's disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD.

Modeling Parkinson's disease genetics: altered function of the dopamine system in Adh4 knockout mice.

Class IV alcohol dehydrogenase (ADH4) efficiently reduces aldehydes produced during lipid peroxidation, and may thus serve to protect from toxic effects of aldehydes e.g. on neurons. We hypothesized that ADH4 dysfunction may increase risk for Parkinson's disease (PD) and previously reported association of an ADH4 allele with PD. We found that a promoter polymorphism in this allele induced a 25-30% reduction of transcriptional activity. Based on these findings, we have now investigated whether Adh4 homo- (Adh4-/-) or heterozygous (Adh4+/-) knockout mice display any dopamine system-related changes in behavior, biochemical parameters or olfaction compared to wild-type mice. The spontaneous locomotor activity was found to be similar in the three groups, whereas administration of d-amphetamine or apomorphine induced a significant increase in horizontal activity in the Adh4-/- mice compared to wild-type mice. We measured levels of monoamines and their metabolites in striatum, frontal cortex and substantia nigra and found increased levels of dopamine and DOPAC in substantia nigra of Adh4-/- mice. Investigation of olfactory function revealed a reduced sense of smell in Adh4-/- mice accompanied by alterations in dopamine metabolite levels in the olfactory bulb. Taken together, our results suggest that lack of Adh4 gene activity induces changes in the function of the dopamine system, findings which are compatible with a role of loss-of-function mutations in ADH4 as possible risk factors for PD.