Metabolic characteristics of transmembrane prolyl 4-hydroxylase (P4H-TM) deficient mice.

Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.

Incidence and Risk Factors of Transient Global Amnesia.

INTRODUCTION: Transient global amnesia (TGA) is a spontaneously resolving, anterograde amnesia that lasts mostly <24 h and often occurs with retrograde amnesia. The etiology of TGA remains unclear, although in recent decades, many risk factors and preceding events have been identified. There are few up-to-date reports on the TGA incidence in Northern Europe. In this study, we report the incidence and risk factors associated with TGA in Finland. MATERIALS AND METHODS: The study included all patients with suspected TGA that were referred to Kuopio University Hospital (KUH) in 2017. The hospital catchment area included 246,653 individuals. Risk factors and demographic data were collected from medical records. The TGA incidence rates were calculated as the number of patients with TGA divided by the number of individuals at risk in different age groups. RESULTS: In 2017, 56 patients were treated for TGA at KUH. Of these, 46 had a first-ever TGA. The most common event preceding TGA was physical effort (n = 28, 50%), followed by emotional stress (n = 11, 19.6%) and water contact or a temperature change (n = 11, 19.6%). The most common comorbidities were hypercholesterolemia (n = 22, 39.3%), hypertensive disease (n = 21, 37.5%), hypothyroidism (n = 11, 19.6%), coronary artery disease (n = 8, 14.3%), and migraine (n = 7, 12.5%). TGA occurred most often in December (n = 9, 16.0%), March (n = 8, 14.3%), or October (n = 8, 14.3%), and least often in November and May (n = 2, 3.6% in both months). The crude incidence of a first TGA in Eastern Finland was 18.6/100,000 inhabitants, and when standardized to the European population in 2010, it was 14.3/100,000 inhabitants. Therefore, the TGA incidence was higher than previously reported in European countries. DISCUSSION: The most common precipitating factors for TGA were physical effort, emotional stress, and water contact/temperature change. The incidence of TGA was high in the Eastern Finnish population.

Activation of the hypoxia response protects mice from amyloid-ß accumulation.

Alzheimer's disease (AD) is the most common cause of dementia with limited treatment options affecting millions of people and the prevalence increasing with the aging population. The current knowledge on the role of the hypoxia/hypoxia-inducible factor (HIF) in the AD pathology is restricted and controversial. We hypothesized based on benefits of the genetic long-term inactivation of HIF prolyl 4-hydroxylase-2 (HIF-P4H-2) on metabolism, vasculature and inflammatory response that prolonged moderate activation of the hypoxia response could hinder AD pathology. We used an aging model to study potential spontaneous accumulation of amyloid-ß (Aß) in HIF-P4H-2-deficient mice and a transgenic APP/PSEN1 mouse model subjected to prolonged sustained environmental hypoxia (15% O2 for 6 weeks) at two different time points of the disease; at age of 4 and 10 months. In both settings, activation of the hypoxia response reduced brain protein aggregate levels and this associated with higher vascularity. In the senescent HIF-P4H-2-deficient mice metabolic reprogramming also contributed to less protein aggregates while in APP/PSEN1 mice lesser Aß associated additionally with hypoxia-mediated favorable responses to neuroinflammation and amyloid precursor protein processing. In conclusion, continuous, non-full-scale activation of the HIF pathway appears to mediate protection against neurodegeneration via several mechanisms and should be studied as a treatment option for AD.

Contribution of astrocytes to familial risk and clinical manifestation of schizophrenia.

Previous studies have implicated several brain cell types in schizophrenia (SCZ), but the genetic impact of astrocytes is unknown. Considering their high complexity in humans, astrocytes are likely key determinants of neurodevelopmental diseases, such as SCZ. Human induced pluripotent stem cell (hiPSC)-derived astrocytes differentiated from five monozygotic twin pairs discordant for SCZ and five healthy subjects were studied for alterations related to high genetic risk and clinical manifestation of SCZ in astrocyte transcriptomics, neuron-astrocyte co-cultures, and in humanized mice. We found gene expression and signaling pathway alterations related to synaptic dysfunction, inflammation, and extracellular matrix components in SCZ astrocytes, and demyelination in SCZ astrocyte transplanted mice. While Ingenuity Pathway Analysis identified SCZ disease and synaptic transmission pathway changes in SCZ astrocytes, the most consistent findings were related to collagen and cell adhesion associated pathways. Neuronal responses to glutamate and GABA differed between astrocytes from control persons, affected twins, and their unaffected co-twins and were normalized by clozapine treatment. SCZ astrocyte cell transplantation to the mouse forebrain caused gene expression changes in synaptic dysfunction and inflammation pathways of mouse brain cells and resulted in behavioral changes in cognitive and olfactory functions. Differentially expressed transcriptomes and signaling pathways related to synaptic functions, inflammation, and especially collagen and glycoprotein 6 pathways indicate abnormal extracellular matrix composition in the brain as one of the key characteristics in the etiology of SCZ.

Whole-brain studies of spontaneous behavior in head-fixed rats enabled by zero echo time MB-SWIFT fMRI.

Understanding the link between the brain activity and behavior is a key challenge in modern neuroscience. Behavioral neuroscience, however, lacks tools to record whole-brain activity in complex behavioral settings. Here we demonstrate that a novel Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT) functional magnetic resonance imaging (fMRI) approach enables whole-brain studies in spontaneously behaving head-fixed rats. First, we show anatomically relevant functional parcellation. Second, we show sensory, motor, exploration, and stress-related brain activity in relevant networks during corresponding spontaneous behavior. Third, we show odor-induced activation of olfactory system with high correlation between the fMRI and behavioral responses. We conclude that the applied methodology enables novel behavioral study designs in rodents focusing on tasks, cognition, emotions, physical exercise, and social interaction. Importantly, novel zero echo time and large bandwidth approaches, such as MB-SWIFT, can be applied for human behavioral studies, allowing more freedom as body movement is dramatically less restricting factor.

Event-recurring multiband SWIFT functional MRI with 200-ms temporal resolution during deep brain stimulation and isoflurane-induced burst suppression in rat.

PURPOSE: To develop a high temporal resolution functional MRI method for tracking repeating events in the brain. METHODS: We developed a novel functional MRI method using multiband sweep imaging with Fourier transformation (SWIFT), termed event-recurring SWIFT (EVER-SWIFT). The method is able to image similar repeating events with subsecond temporal resolution. Here, we demonstrate the use of EVER-SWIFT for detecting functional MRI responses during deep brain stimulation of the medial septal nucleus and during spontaneous isoflurane-induced burst suppression in the rat brain at 9.4 T with 200-ms temporal resolution. RESULTS: The EVER-SWIFT approach showed that the shapes and time-to-peak values of the response curves to deep brain stimulation significantly differed between downstream brain regions connected to the medial septal nucleus, resembling findings obtained with traditional 2-second temporal resolution. In contrast, EVER-SWIFT allowed for detailed temporal measurement of a spontaneous isoflurane-induced bursting activity pattern, which was not achieved with traditional temporal resolution. CONCLUSION: The EVER-SWIFT technique enables subsecond 3D imaging of both stimulated and spontaneously recurring brain activities, and thus holds great potential for studying the mechanisms of neuromodulation and spontaneous brain activity.

Novel Anti-Neuroinflammatory Properties of a Thiosemicarbazone-Pyridylhydrazone Copper(II) Complex.

Neuroinflammation has a major role in several brain disorders including Alzheimer's disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (CuII(gtsm) and CuII(atsm)) have broad therapeutic actions in preclinical models of neurodegeneration, with CuII(atsm) demonstrating beneficial outcomes on neuroinflammatory markers in vitro and in vivo. These findings suggest that copper(II) complexes could be harnessed as a new approach to modulate immune function in neurodegenerative diseases. In this study, we examined the anti-neuroinflammatory action of several low-molecular-weight, charge-neutral and lipophilic copper(II) complexes. Our analysis revealed that one compound, a thiosemicarbazone-pyridylhydrazone copper(II) complex (CuL5), delivered copper into cells in vitro and increased the concentration of copper in the brain in vivo. In a primary murine microglia culture, CuL5 was shown to decrease secretion of pro-inflammatory cytokine macrophage chemoattractant protein 1 (MCP-1) and expression of tumor necrosis factor alpha (Tnf), increase expression of metallothionein (Mt1), and modulate expression of Alzheimer's disease-associated risk genes, Trem2 and Cd33. CuL5 also improved the phagocytic function of microglia in vitro. In 5xFAD model AD mice, treatment with CuL5 led to an improved performance in a spatial working memory test, while, interestingly, increased accumulation of amyloid plaques in treated mice. These findings demonstrate that CuL5 can induce anti-neuroinflammatory effects in vitro and provide selective benefit in vivo. The outcomes provide further support for the development of copper-based compounds to modulate neuroinflammation in brain diseases.

Isoflurane affects brain functional connectivity in rats 1 month after exposure.

Isoflurane, the most commonly used preclinical anesthetic, induces brain plasticity and long-term cellular and molecular changes leading to behavioral and/or cognitive consequences. These changes are most likely associated with network-level changes in brain function. To elucidate the mechanisms underlying long-term effects of isoflurane, we investigated the influence of a single isoflurane exposure on functional connectivity, brain electrical activity, and gene expression. Male Wistar rats (n = 22) were exposed to 1.8% isoflurane for 3 h. Control rats (n = 22) spent 3 h in the same room without exposure to anesthesia. After 1 month, functional connectivity was evaluated with resting-state functional magnetic resonance imaging (fMRI; n = 6 + 6) and local field potential measurements (n = 6 + 6) in anesthetized animals. A whole genome expression analysis (n = 10+10) was also conducted with mRNA-sequencing from cortical and hippocampal tissue samples. Isoflurane treatment strengthened thalamo-cortical and hippocampal-cortical functional connectivity. Cortical low-frequency fMRI power was also significantly increased in response to the isoflurane treatment. The local field potential results indicating strengthened hippocampal-cortical alpha and beta coherence were in good agreement with the fMRI findings. Furthermore, altered expression was found in 20 cortical genes, several of which are involved in neuronal signal transmission, but no gene expression changes were noted in the hippocampus. Isoflurane induced prolonged changes in thalamo-cortical and hippocampal-cortical function and expression of genes contributing to signal transmission in the cortex. Further studies are required to investigate whether these changes are associated with the postoperative behavioral and cognitive symptoms commonly observed in patients and animals.

Deficient astrocyte metabolism impairs glutamine synthesis and neurotransmitter homeostasis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) leads to cerebral accumulation of insoluble amyloid-ß plaques causing synaptic dysfunction and neuronal death. Neurons rely on astrocyte-derived glutamine for replenishment of the amino acid neurotransmitter pools. Perturbations of astrocyte glutamine synthesis have been described in AD, but whether this functionally affects neuronal neurotransmitter synthesis is not known. Since the synthesis and recycling of neurotransmitter glutamate and GABA are intimately coupled to cellular metabolism, the aim of this study was to provide a functional investigation of neuronal and astrocytic energy and neurotransmitter metabolism in AD. To achieve this, we incubated acutely isolated cerebral cortical and hippocampal slices from 8-month-old female 5xFAD mice, in the presence of 13C isotopically enriched substrates, with subsequent gas chromatography-mass spectrometry (GC-MS) analysis. A prominent neuronal hypometabolism of [U-13C]glucose was observed in the hippocampal slices of the 5xFAD mice. Investigating astrocyte metabolism, using [1,2-13C]acetate, revealed a marked reduction in glutamine synthesis, which directly hampered neuronal synthesis of GABA. This was supported by an increased metabolism of exogenously supplied [U-13C]glutamine, suggesting a neuronal metabolic compensation of the reduced astrocytic glutamine supply. In contrast, astrocytic metabolism of [U-13C]GABA was reduced, whereas [U-13C]glutamate metabolism was unaffected. Finally, astrocyte de novo synthesis of glutamate and glutamine was hampered, whereas the enzymatic capacity of glutamine synthetase for ammonia fixation was maintained. Collectively, we demonstrate that deficient astrocyte metabolism leads to reduced glutamine synthesis, directly impairing neuronal GABA synthesis in the 5xFAD brain. These findings suggest that astrocyte metabolic dysfunction may be fundamental for the imbalances of synaptic excitation and inhibition in the AD brain.

Loss of Cln5 leads to altered Gad1 expression and deficits in interneuron development in mice.

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5-/- embryos of various ages and cells harvested from Cln5-/- brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5-/- mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5-/- embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5-/- mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

Neuronal spreading and plaque induction of intracellular Aß and its disruption of Aß homeostasis.

The amyloid-beta peptide (Aß) is thought to have prion-like properties promoting its spread throughout the brain in Alzheimer's disease (AD). However, the cellular mechanism(s) of this spread remains unclear. Here, we show an important role of intracellular Aß in its prion-like spread. We demonstrate that an intracellular source of Aß can induce amyloid plaques in vivo via hippocampal injection. We show that hippocampal injection of mouse AD brain homogenate not only induces plaques, but also damages interneurons and affects intracellular Aß levels in synaptically connected brain areas, paralleling cellular changes seen in AD. Furthermore, in a primary neuron AD model, exposure of picomolar amounts of brain-derived Aß leads to an apparent redistribution of Aß from soma to processes and dystrophic neurites. We also observe that such neuritic dystrophies associate with plaque formation in AD-transgenic mice. Finally, using cellular models, we propose a mechanism for how intracellular accumulation of Aß disturbs homeostatic control of Aß levels and can contribute to the up to 10,000-fold increase of Aß in the AD brain. Our data indicate an essential role for intracellular prion-like Aß and its synaptic spread in the pathogenesis of AD.

Multi-band SWIFT enables quiet and artefact-free EEG-fMRI and awake fMRI studies in rat.

Functional magnetic resonance imaging (fMRI) studies in animal models provide invaluable information regarding normal and abnormal brain function, especially when combined with complementary stimulation and recording techniques. The echo planar imaging (EPI) pulse sequence is the most common choice for fMRI investigations, but it has several shortcomings. EPI is one of the loudest sequences and very prone to movement and susceptibility-induced artefacts, making it suboptimal for awake imaging. Additionally, the fast gradient-switching of EPI induces disrupting currents in simultaneous electrophysiological recordings. Therefore, we investigated whether the unique features of Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT) overcome these issues at a high 9.4 T magnetic field, making it a potential alternative to EPI. MB-SWIFT had 32-dB and 20-dB lower peak and average sound pressure levels, respectively, than EPI with typical fMRI parameters. Body movements had little to no effect on MB-SWIFT images or functional connectivity analyses, whereas they severely affected EPI data. The minimal gradient steps of MB-SWIFT induced significantly lower currents in simultaneous electrophysiological recordings than EPI, and there were no electrode-induced distortions in MB-SWIFT images. An independent component analysis of the awake rat functional connectivity data obtained with MB-SWIFT resulted in near whole-brain level functional parcellation, and simultaneous electrophysiological and fMRI measurements in isoflurane-anesthetized rats indicated that MB-SWIFT signal is tightly linked to neuronal resting-state activity. Therefore, we conclude that the MB-SWIFT sequence is a robust preclinical brain mapping tool that can overcome many of the drawbacks of conventional EPI fMRI at high magnetic fields.

Orientation selective deep brain stimulation of the subthalamic nucleus in rats.

Deep brain stimulation (DBS) has become an important tool in the management of a wide spectrum of diseases in neurology and psychiatry. Target selection is a vital aspect of DBS so that only the desired areas are stimulated. Segmented leads and current steering have been shown to be promising additions to DBS technology enabling better control of the stimulating electric field. Recently introduced orientation selective DBS (OS-DBS) is a related development permitting sensitization of the stimulus to axonal pathways with different orientations by freely controlling the primary direction of the electric field using multiple contacts. Here, we used OS-DBS to stimulate the subthalamic nucleus (STN) in healthy rats while simultaneously monitoring the induced brain activity with fMRI. Maximal activation of the sensorimotor and basal ganglia-thalamocortical networks was observed when the electric field was aligned mediolaterally in the STN pointing in the lateral direction, while no cortical activation was observed with the electric field pointing medially to the opposite direction. Such findings are consistent with mediolateral main direction of the STN fibers, as seen with high resolution diffusion imaging and histology. The asymmetry of the OS-DBS dipolar field distribution using three contacts along with the potential stimulation of the internal capsule, are also discussed. We conclude that OS-DBS offers an additional degree of flexibility for optimization of DBS of the STN which may enable a better treatment response.

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease.

BACKGROUND: FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. METHODS: The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. RESULTS: Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. CONCLUSIONS: We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

Astrocyte remodeling in the beneficial effects of long-term voluntary exercise in Alzheimer's disease.

BACKGROUND: Increased physical exercise improves cognitive function and reduces pathology associated with Alzheimer's disease (AD). However, the mechanisms underlying the beneficial effects of exercise in AD on the level of specific brain cell types remain poorly investigated. The involvement of astrocytes in AD pathology is widely described, but their exact role in exercise-mediated neuroprotection warrant further investigation. Here, we investigated the effect of long-term voluntary physical exercise on the modulation of the astrocyte state. METHODS: Male 5xFAD mice and their wild-type littermates had free access to a running wheel from 1.5 to 7 months of age. A battery of behavioral tests was used to assess the effects of voluntary exercise on cognition and learning. Neuronal loss, impairment in neurogenesis, beta-amyloid (Aß) deposition, and inflammation were evaluated using a variety of histological and biochemical measurements. Sophisticated morphological analyses were performed to delineate the specific involvement of astrocytes in exercise-induced neuroprotection in the 5xFAD mice. RESULTS: Long-term voluntary physical exercise reversed cognitive impairment in 7-month-old 5xFAD mice without affecting neurogenesis, neuronal loss, Aß plaque deposition, or microglia activation. Exercise increased glial fibrillary acid protein (GFAP) immunoreactivity and the number of GFAP-positive astrocytes in 5xFAD hippocampi. GFAP-positive astrocytes in hippocampi of the exercised 5xFAD mice displayed increases in the numbers of primary branches and in the soma area. In general, astrocytes distant from Aß plaques were smaller in size and possessed simplified processes in comparison to plaque-associated GFAP-positive astrocytes. Morphological alterations of GFAP-positive astrocytes occurred concomitantly with increased astrocytic brain-derived neurotrophic factor (BDNF) and restoration of postsynaptic protein PSD-95. CONCLUSIONS: Voluntary physical exercise modulates the reactive astrocyte state, which could be linked via astrocytic BDNF and PSD-95 to improved cognition in 5xFAD hippocampi. The molecular pathways involved in this modulation could potentially be targeted for benefit against AD.

Impaired Mitochondrial Fatty Acid Synthesis Leads to Neurodegeneration in Mice.

There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties ∼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.

Collagen XIII Is Required for Neuromuscular Synapse Regeneration and Functional Recovery after Peripheral Nerve Injury.

Collagen XIII occurs as both a transmembrane-bound and a shed extracellular protein and is able to regulate the formation and function of neuromuscular synapses. Its absence results in myasthenia: presynaptic and postsynaptic defects at the neuromuscular junction (NMJ), leading to destabilization of the motor nerves, muscle regeneration and atrophy. Mutations in COL13A1 have recently been found to cause congenital myasthenic syndrome, characterized by fatigue and chronic muscle weakness, which may be lethal. We show here that muscle defects in collagen XIII-deficient mice stabilize in adulthood, so that the disease is not progressive until very late. Sciatic nerve crush was performed to examine how the lack of collagen XIII or forced expression of its transmembrane form affects the neuromuscular synapse regeneration and functional recovery following injury. We show that collagen XIII-deficient male mice are unable to achieve complete NMJ regeneration and functional recovery. This is mainly attributable to presynaptic defects that already existed in the absence of collagen XIII before injury. Shedding of the ectodomain is not required, as the transmembrane form of collagen XIII alone fully rescues the phenotype. Thus, collagen XIII could serve as a therapeutic agent in cases of injury-induced PNS regeneration and functional recovery. We conclude that intrinsic alterations at the NMJ in Col13a1-/- mice contribute to impaired and incomplete NMJ regeneration and functional recovery after peripheral nerve injury. However, such alterations do not progress once they have stabilized in early adulthood, emphasizing the role of collagen XIII in NMJ maturation.SIGNIFICANCE STATEMENT Collagen XIII is required for gaining and maintaining the normal size, complexity, and functional capacity of neuromuscular synapses. Loss-of-function mutations in COL13A1 cause congenital myasthenic syndrome 19, characterized by postnatally progressive muscle fatigue, which compromises patients' functional capacity. We show here in collagen XIII-deficient mice that the disease stabilizes in adulthood once the NMJs have matured. This study also describes a relevant contribution of the altered NMJ morphology and function to neuromuscular synapses, and PNS regeneration and functional recovery in collagen XIII-deficient mice after peripheral nerve injury. Correlating the animal model data on collagen XIII-associated congenital myasthenic syndrome, it can be speculated that neuromuscular connections in congenital myasthenic syndrome patients are not able to fully regenerate and restore normal functionality if exposed to peripheral nerve injury.

PPARß/δ-agonist GW0742 ameliorates dysfunction in fatty acid oxidation in PSEN1ΔE9 astrocytes.

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bioenergetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer's disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARß/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aß) load, GW0742 treatment increased hippocampal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aß-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARß/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.

Lack of P4H-TM in mice results in age-related retinal and renal alterations.

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm-/- mice were fertile and had a normal life span. Lack of P4h-tm stabilized HIF-1α in cortical neurons under normoxia, while in hypoxia it increased the expression of certain HIF target genes in tissues with high endogenous P4h-tm expression levels more than in wild-type mice. Renal erythropoietin levels increased in P4h-tm-/- mice with aging, but the resulting ∼2-fold increase in erythropoietin serum levels did not lead to erythrocytosis. Instead, accumulation of lipid-containing lamellar bodies in renal tubuli was detected in P4h-tm-/- mice with aging, resulting in inflammation and fibrosis, and later glomerular sclerosis and albuminuria. Lack of P4h-tm was associated with retinal thinning, rosette-like infoldings and drusen-like structure accumulation in RPE with aging, as is characteristic of AMD. Photoreceptor recycling was compromised, and electroretinograms revealed functional impairment of the cone pathway in adult P4h-tm-/- mice and cone and rod deficiency in middle-aged mice. P4H-TM is therefore imperative for normal vision, and potentially a novel candidate for age-induced diseases, such as AMD.

SEPT8 modulates ß-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.

Dysfunction and loss of synapses are early pathogenic events in Alzheimer's disease. A central step in the generation of toxic amyloid-ß (Aß) peptides is the cleavage of amyloid precursor protein (APP) by ß-site APP-cleaving enzyme (BACE1). Here, we have elucidated whether downregulation of septin (SEPT) protein family members, which are implicated in synaptic plasticity and vesicular trafficking, affects APP processing and Aß generation. SEPT8 was found to reduce soluble APPß and Aß levels in neuronal cells through a post-translational mechanism leading to decreased levels of BACE1 protein. In the human temporal cortex, we identified alterations in the expression of specific SEPT8 transcript variants in a manner that correlated with Alzheimer's-disease-related neurofibrillary pathology. These changes were associated with altered ß-secretase activity. We also discovered that the overexpression of a specific Alzheimer's-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aß peptides in neuronal cells. These changes were related to an increased half-life of BACE1 and the localization of BACE1 in recycling endosomes. These data suggest that SEPT8 modulates ß-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1.