A ketogenic intervention improves dorsal attention network functional and structural connectivity in mild cognitive impairment.

Ketones, the brain's alternative fuel to glucose, bypass the brain glucose deficit and improve cognition in mild cognitive impairment (MCI). Our goal was to assess the impact of a 6-month ketogenic intervention on the functional connectivity within eight major brain resting-state networks, and its possible relationship to improved cognitive outcomes in the BENEFIC trial. MCI participants were randomized to a placebo (n = 15) or ketogenic medium chain triglyceride (kMCT; n = 17) intervention. kMCT was associated with increased functional connectivity within the dorsal attention network (DAN), which correlated to improvement in cognitive tests targeting attention. Ketone uptake (11C-acetoacetate PET) specifically in DAN cortical regions was highly increased in the kMCT group and was directly associated with the improved DAN functional connectivity. Analysis of the structural connectome revealed increased fiber density within the DAN following kMCT. Our findings suggest that ketones in MCI may prove beneficial for cognition at least in part because they improve brain network energy status, functional connectivity and axonal integrity.

Tractostorm 2: Optimizing tractography dissection reproducibility with segmentation protocol dissemination.

The segmentation of brain structures is a key component of many neuroimaging studies. Consistent anatomical definitions are crucial to ensure consensus on the position and shape of brain structures, but segmentations are prone to variation in their interpretation and execution. White-matter (WM) pathways are global structures of the brain defined by local landmarks, which leads to anatomical definitions being difficult to convey, learn, or teach. Moreover, the complex shape of WM pathways and their representation using tractography (streamlines) make the design and evaluation of dissection protocols difficult and time-consuming. The first iteration of Tractostorm quantified the variability of a pyramidal tract dissection protocol and compared results between experts in neuroanatomy and nonexperts. Despite virtual dissection being used for decades, in-depth investigations of how learning or practicing such protocols impact dissection results are nonexistent. To begin to fill the gap, we evaluate an online educational tractography course and investigate the impact learning and practicing a dissection protocol has on interrater (groupwise) reproducibility. To generate the required data to quantify reproducibility across raters and time, 20 independent raters performed dissections of three bundles of interest on five Human Connectome Project subjects, each with four timepoints. Our investigation shows that the dissection protocol in conjunction with an online course achieves a high level of reproducibility (between 0.85 and 0.90 for the voxel-based Dice score) for the three bundles of interest and remains stable over time (repetition of the protocol). Suggesting that once raters are familiar with the software and tasks at hand, their interpretation and execution at the group level do not drastically vary. When compared to previous work that used a different method of communication for the protocol, our results show that incorporating a virtual educational session increased reproducibility. Insights from this work may be used to improve the future design of WM pathway dissection protocols and to further inform neuroanatomical definitions.

A ketogenic supplement improves white matter energy supply and processing speed in mild cognitive impairment.

INTRODUCTION: White matter (WM) energy supply is crucial for axonal function and myelin maintenance. An exogenous source of ketones, the brain's alternative fuel to glucose, bypasses the brain's glucose-specific energy deficit and improves cognitive outcomes in mild cognitive impairment (MCI). How an additional supply of ketones affects glucose or ketone uptake in specific WM fascicles in MCI has not previously been reported. METHODS: This 6-month interventional study included MCI participants randomized to a placebo (n = 16) or ketogenic medium chain triglyceride (kMCT; n = 17) drink. A neurocognitive battery and brain imaging were performed pre- and post-intervention. WM fascicle uptake of ketone and glucose and structural properties were assessed using positron emission tomography and diffusion imaging, respectively. RESULTS: Ketone uptake was increased in the kMCT group by 2.5- to 3.2-fold in all nine WM fascicles of interest (P < .001), an effect seen both in deep WM and in fascicle cortical endpoints. Improvement in processing speed was positively associated with WM ketone uptake globally and in individual fascicles, most importantly the fornix (r = +0.61; P = .014). DISCUSSION: A 6-month kMCT supplement improved WM energy supply in MCI by increasing ketone uptake in WM fascicles. The significant positive association with processing speed suggests that ketones may have a role in myelin integrity in MCI.

Bundle-specific associations between white matter microstructure and Aß and tau pathology in preclinical Alzheimer's disease.

Beta-amyloid (Aß) and tau proteins, the pathological hallmarks of Alzheimer's disease (AD), are believed to spread through connected regions of the brain. Combining diffusion imaging and positron emission tomography, we investigated associations between white matter microstructure specifically in bundles connecting regions where Aß or tau accumulates and pathology. We focused on free-water-corrected diffusion measures in the anterior cingulum, posterior cingulum, and uncinate fasciculus in cognitively normal older adults at risk of sporadic AD and presymptomatic mutation carriers of autosomal dominant AD. In Aß-positive or tau-positive groups, lower tissue fractional anisotropy and higher mean diffusivity related to greater Aß and tau burden in both cohorts. Associations were found in the posterior cingulum and uncinate fasciculus in preclinical sporadic AD, and in the anterior and posterior cingulum in presymptomatic mutation carriers. These results suggest that microstructural alterations accompany pathological accumulation as early as the preclinical stage of both sporadic and autosomal dominant AD.

A ketogenic drink improves cognition in mild cognitive impairment: Results of a 6-month RCT.

INTRODUCTION: Counteracting impaired brain glucose metabolism with ketones may improve cognition in mild cognitive impairment (MCI). METHODS: Cognition, plasma ketone response, and metabolic profile were assessed before and 6 months after supplementation with a ketogenic drink containing medium chain triglyceride (ketogenic medium chain triglyceride [kMCT]; 15 g twice/day; n = 39) or placebo (n = 44). RESULTS: Free and cued recall (Trial 1; P = .047), verbal fluency (categories; P = .024), Boston Naming Test (total correct answers; P = .033), and the Trail-Making Test (total errors; P = .017) improved significantly in the kMCT group compared to placebo (analysis of covariance; pre-intervention score, sex, age, education, and apolipoprotein E4 as covariates). Some cognitive outcomes also correlated positively with plasma ketones. Plasma metabolic profile and ketone response were unchanged. CONCLUSIONS: This kMCT drink improved cognitive outcomes in MCI, at least in part by increasing blood ketone level. These data support further assessment of MCI progression to Alzheimer's disease.

Fascicle- and Glucose-Specific Deterioration in White Matter Energy Supply in Alzheimer's Disease.

BACKGROUND: White matter energy supply to oligodendrocytes and the axonal compartment is crucial for normal axonal function. Although gray matter glucose hypometabolism is extensively reported in Alzheimer's disease (AD), glucose and ketones, the brain's two main fuels, are rarely quantified in white matter in AD. OBJECTIVE: Using a dual-tracer PET method combined with a fascicle-specific diffusion MRI approach, robust to white matter hyper intensities and crossing fibers, we aimed to quantify both glucose and ketone metabolism in specific white matter fascicles associated with mild cognitive impairment (MCI; n = 51) and AD (n = 13) compared to cognitively healthy age-matched controls (Controls; n = 14). METHODS: Eight white matter fascicles of the limbic lobe and corpus callosum were extracted and analyzed into fascicle profiles of five sections. Glucose (18F-fluorodeoxyglucose) and ketone (11C-acetoacetate) uptake rates, corrected for partial volume effect, were calculated along each fascicle. RESULTS: The only fascicle with significantly lower glucose uptake in AD compared to Controls was the left posterior cingulate segment of the cingulum (-22%; p = 0.016). Non-significantly lower glucose uptake in this fascicle was also observed in MCI. In contrast to glucose, ketone uptake was either unchanged or higher in sections of the fornix and parahippocampal segment of the cingulum in AD. CONCLUSION: To our knowledge, this is the first report of brain fuel uptake calculated along white matter fascicles in humans. Energetic deterioration in white matter in AD appears to be specific to glucose and occurs first in the posterior cingulum.

Free Water in White Matter Differentiates MCI and AD From Control Subjects.

Recent evidence shows that neuroinflammation plays a role in many neurological diseases including mild cognitive impairment (MCI) and Alzheimer's disease (AD), and that free water (FW) modeling from clinically acquired diffusion MRI (DTI-like acquisitions) can be sensitive to this phenomenon. This FW index measures the fraction of the diffusion signal explained by isotropically unconstrained water, as estimated from a bi-tensor model. In this study, we developed a simple but powerful whole-brain FW measure designed for easy translation to clinical settings and potential use as a priori outcome measure in clinical trials. These simple FW measures use a "safe" white matter (WM) mask without gray matter (GM)/CSF partial volume contamination (WM safe) near ventricles and sulci. We investigated if FW inside the WM safe mask, including and excluding areas of white matter damage such as white matter hyperintensities (WMHs) as shown on T2 FLAIR, computed across the whole white matter could be indicative of diagnostic grouping along the AD continuum. After careful quality control, 81 cognitively normal controls (NC), 103 subjects with MCI and 42 with AD were selected from the ADNIGO and ADNI2 databases. We show that MCI and AD have significantly higher FW measures even after removing all partial volume contamination. We also show, for the first time, that when WMHs are removed from the masks, the significant results are maintained, which demonstrates that the FW measures are not just a byproduct of WMHs. Our new and simple FW measures can be used to increase our understanding of the role of inflammation-associated edema in AD and may aid in the differentiation of healthy subjects from MCI and AD patients.

A ketogenic drink improves brain energy and some measures of cognition in mild cognitive impairment.

INTRODUCTION: Unlike for glucose, uptake of the brain's main alternative fuel, ketones, remains normal in mild cognitive impairment (MCI). Ketogenic medium chain triglycerides (kMCTs) could improve cognition in MCI by providing the brain with more fuel. METHODS: Fifty-two subjects with MCI were blindly randomized to 30 g/day of kMCT or matching placebo. Brain ketone and glucose metabolism (quantified by positron emission tomography; primary outcome) and cognitive performance (secondary outcome) were assessed at baseline and 6 months later. RESULTS: Brain ketone metabolism increased by 230% for subjects on the kMCT (P < .001) whereas brain glucose uptake remained unchanged. Measures of episodic memory, language, executive function, and processing speed improved on the kMCT versus baseline. Increased brain ketone uptake was positively related to several cognitive measures. Seventy-five percent of participants completed the intervention. DISCUSSION: A dose of 30 g/day of kMCT taken for 6 months bypasses a significant part of the brain glucose deficit and improves several cognitive outcomes in MCI.

Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using (1)H- and (13)C-NMR spectroscopy.

The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-(13)C]glucose (Glc) or [2,4-(13)C2]ß-hydroxybutyrate (ß-HB). Specific enrichments of amino acids (AAs) measured by (1)H- and (13)C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-(13)C]Glc infusion whereas they were higher in KD rats after [2,4-(13)C2]ß-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.

Energy restriction does not prevent insulin resistance but does prevent liver steatosis in aging rats on a Western-style diet.

OBJECTIVE: The aim of this study was to evaluate the effects of long-term energy restriction (ER) on plasma, liver, and skeletal muscle metabolite profiles in aging rats fed a Western-style diet. METHODS: Three groups of male Sprague-Dawley rats were studied. Group 1 consisted of 2 mo old rats fed ad libitum; group 2 were 19 mo old rats also fed ad libitum; and group 3 were 19 mo old rats subjected to 40% ER for the last 11.5 mo. To imitate a Western-style diet, all rats were given a high-sucrose, very low ω-3 polyunsaturated fatty acid (PUFA) diet. High-resolution magic angle spinning-(1)H nuclear magnetic resonance spectroscopy was used for hepatic and skeletal muscle metabolite determination, and fatty acid profiles were measured by capillary gas chromatography on plasma, liver, and skeletal muscle. RESULTS: ER coupled with a Western-style diet did not prevent age-induced insulin resistance or the increase in triacylglycerol content in plasma and skeletal muscle associated with aging. However, in the liver, ER did prevent steatosis and increased the percent of saturated and monounsaturated fatty acids relative to ω-6 and ω-3 PUFA. CONCLUSIONS: Although steatosis was reduced, the beneficial effects of ER on systemic insulin resistance and plasma and skeletal muscle metabolites observed elsewhere with a balanced diet seem to be compromised by high-sucrose and low ω-3 PUFA intake.

Age-associated evolution of plasmatic amyloid in mouse lemur primates: relationship with intracellular amyloid deposition.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Amyloid-ß peptide (Aß) deposition in the brain is one of its hallmarks, and the measure of plasma Aß is considered to be a biomarker for anti-amyloid drug efficacy in animal models of AD. However, age-associated plasmatic Aß modulation in animal models is practically never addressed in the literature. Mouse lemur primates are used as a model of normal and AD-like cerebral aging. Here, we studied the effect of age on plasmatic Aß in 58 mouse lemurs aged from 1 to 10 years. A subset of animals presented high plasmatic Aß, and the proportion of animals with high plasmatic Aß was higher in aged animals as compared with young ones. Histologic evaluation of the brain of some of these animals was carried out to assess extracellular and intracellular amyloid load. In aged lemurs, plasmatic Aß was negatively correlated with the density of neurons accumulating deposits of Aß.

Brain glucose and acetoacetate metabolism: a comparison of young and older adults.

The extent to which the age-related decline in regional brain glucose uptake also applies to other important brain fuels is presently unknown. Ketones are the brain's major alternative fuel to glucose, so we developed a dual tracer positron emission tomography protocol to quantify and compare regional cerebral metabolic rates for glucose and the ketone, acetoacetate. Twenty healthy young adults (mean age, 26 years) and 24 healthy older adults (mean age, 74 years) were studied. In comparison with younger adults, older adults had 8 ± 6% (mean ± SD) lower cerebral metabolic rates for glucose in gray matter as a whole (p = 0.035), specifically in several frontal, temporal, and subcortical regions, as well as in the cingulate and insula (p ≤ 0.01, false discovery rate correction). The effect of age on cerebral metabolic rates for acetoacetate in gray matter did not reach significance (p = 0.11). Rate constants (min(-1)) of glucose (Kg) and acetoacetate (Ka) were significantly lower (-11 ± 6%; [p = 0.005], and -19 ± 5%; [p = 0.006], respectively) in older adults compared with younger adults. There were differential effects of age on Kg and Ka as seen by significant interaction effects in the caudate (p = 0.030) and post-central gyrus (p = 0.023). The acetoacetate index, which expresses the scaled residuals of the voxel-wise linear regression of glucose on ketone uptake, identifies regions taking up higher or lower amounts of acetoacetate relative to glucose. The acetoacetate index was higher in the caudate of young adults when compared with older adults (p ≤ 0.05 false discovery rate correction). This study provides new information about glucose and ketone metabolism in the human brain and a comparison of the extent to which their regional use changes during normal aging.

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet.

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague-Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-(1)H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), ß-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35-47% lower but monounsaturated fatty acids were 40-52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.

Stimulation of mild, sustained ketonemia by medium-chain triacylglycerols in healthy humans: estimated potential contribution to brain energy metabolism.

OBJECTIVE: In humans consuming a normal diet, we investigated 1) the capacity of a medium-chain triacylglycerol (MCT) supplement to stimulate and sustain ketonemia, 2) ¹³C-ß-hydroxybutyrate and ¹³C-trioctanoate metabolism, and 3) the theoretical contribution of the degree of ketonemia achieved to brain energy metabolism. METHODS: Eight healthy adults (26 ± 1 y old) were given an MCT supplement for 4 wk (4 times/d; total of 20 g/d for 1 wk followed by 30 g/d for 3 wk). Ketones, glucose, triacylglycerols, cholesterol, free fatty acids, and insulin were measured over 8 h during two separate metabolic study days before and after MCT supplementation. Using isotope ratio mass spectroscopy, ¹³C-D-ß-hydroxybutyrate and ¹³C-trioctanoate ß-oxidation to ¹³CO2 was measured over 12 h on the pre- and post-MCT metabolic study days. RESULTS: On the post-MCT metabolic study day, plasma ketones (ß-hydroxybutyrate plus acetoacetate) peaked at 476 µM, with a mean value throughout the study day of 290 µM. Post-MCT, ¹³C-trioctanoate ß-oxidation was significantly lower 1 to 8 h later but higher 10 to 12 h later. MCT supplementation did not significantly alter ¹³C-D-ß-hydroxybutyrate oxidation. CONCLUSIONS: This MCT supplementation protocol was mildly and safely ketogenic and had no side effects in healthy humans on their regular diet. This degree of ketonemia is estimated to contribute up to 8% to 9% of brain energy metabolism.

A dual tracer PET-MRI protocol for the quantitative measure of regional brain energy substrates uptake in the rat.

We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which (11)C-AcAc and (18)F-fluorodeoxyglucose ((18)F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of (11)C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of (11)C-AcAc and (18)F-FDG brain uptake (cerebral metabolic rate; µmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.

The ketogenic diet increases brain glucose and ketone uptake in aged rats: a dual tracer PET and volumetric MRI study.

Despite decades of study, it is still unclear whether regional brain glucose uptake is lower in the cognitively healthy elderly. Whether regional brain uptake of ketones (ß-hydroxybutyrate and acetoacetate [AcAc]), the main alternative brain fuel to glucose, changes with age is unknown. We used a sequential, dual tracer positron emission tomography (PET) protocol to quantify brain (18)F-fluorodeoxyglucose ((18)F-FDG) and (11)C-AcAc uptake in two studies with healthy, male Sprague-Dawley rats: (i) Aged (21 months; 21M) versus young (4 months; 4M) rats, and (ii) The effect of a 14 day high-fat ketogenic diet (KD) on brain (18)F-FDG and (11)C-AcAc uptake in 24 month old rats (24M). Similar whole brain volumes assessed by magnetic resonance imaging, were observed in aged 21M versus 4M rats, but the lateral ventricles were 30% larger in the 21M rats (p=0.001). Whole brain cerebral metabolic rates of AcAc (CMR(AcAc)) and glucose (CMR(glc)) did not differ between 21M and 4M rats, but were 28% and 44% higher, respectively, in 24M-KD compared to 24M rats. The region-to-whole brain ratio of CMR(glc) was 37-41% lower in the cortex and 40-45% lower in the cerebellum compared to CMR(AcAc) in 4M and 21M rats. We conclude that a quantitative measure of uptake of the brain's two principal exogenous fuels was generally similar in healthy aged and young rats, that the % of distribution across brain regions differed between ketones and glucose, and that brain uptake of both fuels was stimulated by mild, experimental ketonemia.

γ-Secretase-mediated regulation of neprilysin: influence of cell density and aging and modulation by imatinib.

Proteolytic degradation has emerged as a key pathway involved in controlling levels of the Alzheimer's disease (AD)-associated amyloid-ß peptides (Aß) in the brain. The ectopeptidase, neprilysin (NEP), has been reported as the major Aß-degrading enzyme in mice and human brains. We have previously shown that NEP expression and activity are regulated by AICD, the intracellular domain of the amyloid-ß protein precursor (AßPP) generated by γ-secretase. Thus, NEP transcription, expression, and enzymatic activity are dramatically reduced in fibroblasts devoid of AßPP (the precursor of AICD) or lacking both presenilin (PS) 1 and 2 (two parent proteins contributing to AICD formation). We demonstrate here that NEP expression and activity are influenced by a number of cell passages and density, and we confirm a drastic reduction of NEP expression and activity in AßPP and PS null fibroblasts examined at similar passages and cell densities. Furthermore, Imatinib (Gleevec), a known tyrosine kinase inhibitor was recently shown to elevate AICD in H4 human neuroglioma cells, and this was accompanied by concomitant increases of NEP protein, mRNA levels, and activity. However, the demonstration of a causal link between Imatinib and AICD levels was still lacking. We show here an Imatinib-dependent effect on NEP expression and activity in murine fibroblasts and establish that Imatinib-induced modulation of NEP was abolished by the depletion of AßPP or its homologues APLP1 and APLP2, thereby confirming that Imatinib-mediated control of NEP could indeed be accounted for its effect on AICD.

Brain fuel metabolism, aging, and Alzheimer's disease.

Lower brain glucose metabolism is present before the onset of clinically measurable cognitive decline in two groups of people at risk of Alzheimer's disease--carriers of apolipoprotein E4, and in those with a maternal family history of AD. Supported by emerging evidence from in vitro and animal studies, these reports suggest that brain hypometabolism may precede and therefore contribute to the neuropathologic cascade leading to cognitive decline in AD. The reason brain hypometabolism develops is unclear but may include defects in brain glucose transport, disrupted glycolysis, and/or impaired mitochondrial function. Methodologic issues presently preclude knowing with certainty whether or not aging in the absence of cognitive impairment is necessarily associated with lower brain glucose metabolism. Nevertheless, aging appears to increase the risk of deteriorating systemic control of glucose utilization, which, in turn, may increase the risk of declining brain glucose uptake, at least in some brain regions. A contributing role of deteriorating glucose availability to or metabolism by the brain in AD does not exclude the opposite effect, i.e., that neurodegenerative processes in AD further decrease brain glucose metabolism because of reduced synaptic functionality and hence reduced energy needs, thereby completing a vicious cycle. Strategies to reduce the risk of AD by breaking this cycle should aim to (1) improve insulin sensitivity by improving systemic glucose utilization, or (2) bypass deteriorating brain glucose metabolism using approaches that safely induce mild, sustainable ketonemia.