Left frontal hub connectivity delays cognitive impairment in autosomal-dominant and sporadic Alzheimer\u27s disease

Patients with Alzheimer\u27s disease vary in their ability to sustain cognitive abilities in the presence of brain pathology. A major open question is which brain mechanisms may support higher reserve capacity, i.e. relatively high cognitive performance at a given level of Alzheimer\u27s pathology. Higher functional MRI-assessed functional connectivity of a hub in the left frontal cortex is a core candidate brain mechanism underlying reserve as it is associated with education (i.e. a protective factor often associated with higher reserve) and attenuated cognitive impairment in prodromal Alzheimer\u27s disease. However, no study has yet assessed whether such hub connectivity of the left frontal cortex supports reserve throughout the evolution of pathological brain changes in Alzheimer\u27s disease, including the presymptomatic stage when cognitive decline is subtle. To address this research gap, we obtained cross-sectional resting state functional MRI in 74 participants with autosomal dominant Alzheimer\u27s disease, 55 controls from the Dominantly Inherited Alzheimer\u27s Network and 75 amyloid-positive elderly participants, as well as 41 amyloid-negative cognitively normal elderly subjects from the German Center of Neurodegenerative Diseases multicentre study on biomarkers in sporadic Alzheimer\u27s disease. For each participant, global left frontal cortex connectivity was computed as the average resting state functional connectivity between the left frontal cortex (seed) and each voxel in the grey matter. As a marker of disease stage, we applied estimated years from symptom onset in autosomal dominantly inherited Alzheimer\u27s disease and cerebrospinal fluid tau levels in sporadic Alzheimer\u27s disease cases. In both autosomal dominant and sporadic Alzheimer\u27s disease patients, higher levels of left frontal cortex connectivity were correlated with greater education. For autosomal dominant Alzheimer\u27s disease, a significant left frontal cortex connectivity × estimated years of onset interaction was found, indicating slower decline of memory and global cognition at higher levels of connectivity. Similarly, in sporadic amyloid-positive elderly subjects, the effect of tau on cognition was attenuated at higher levels of left frontal cortex connectivity. Polynomial regression analysis showed that the trajectory of cognitive decline was shifted towards a later stage of Alzheimer\u27s disease in patients with higher levels of left frontal cortex connectivity. Together, our findings suggest that higher resilience against the development of cognitive impairment throughout the early stages of Alzheimer\u27s disease is at least partially attributable to higher left frontal cortex-hub connectivity

Left frontal hub connectivity delays cognitive impairment in autosomal-dominant and sporadic Alzheimer's disease

Patients with Alzheimer's disease vary in their ability to sustain cognitive abilities in the presence of brain pathology. A major open question is which brain mechanisms may support higher reserve capacity, i.e. relatively high cognitive performance at a given level of Alzheimer's pathology. Higher functional MRI-assessed functional connectivity of a hub in the left frontal cortex is a core candidate brain mechanism underlying reserve as it is associated with education (i.e. a protective factor often associated with higher reserve) and attenuated cognitive impairment in prodromal Alzheimer's disease. However, no study has yet assessed whether such hub connectivity of the left frontal cortex supports reserve throughout the evolution of pathological brain changes in Alzheimer's disease, including the presymptomatic stage when cognitive decline is subtle. To address this research gap, we obtained cross-sectional resting state functional MRI in 74 participants with autosomal dominant Alzheimer's disease, 55 controls from the Dominantly Inherited Alzheimer's Network and 75 amyloid-positive elderly participants, as well as 41 amyloid-negative cognitively normal elderly subjects from the German Center of Neurodegenerative Diseases multicentre study on biomarkers in sporadic Alzheimer's disease. For each participant, global left frontal cortex connectivity was computed as the average resting state functional connectivity between the left frontal cortex (seed) and each voxel in the grey matter. As a marker of disease stage, we applied estimated years from symptom onset in autosomal dominantly inherited Alzheimer's disease and cerebrospinal fluid tau levels in sporadic Alzheimer's disease cases. In both autosomal dominant and sporadic Alzheimer's disease patients, higher levels of left frontal cortex connectivity were correlated with greater education. For autosomal dominant Alzheimer's disease, a significant left frontal cortex connectivity × estimated years of onset interaction was found, indicating slower decline of memory and global cognition at higher levels of connectivity. Similarly, in sporadic amyloid-positive elderly subjects, the effect of tau on cognition was attenuated at higher levels of left frontal cortex connectivity. Polynomial regression analysis showed that the trajectory of cognitive decline was shifted towards a later stage of Alzheimer's disease in patients with higher levels of left frontal cortex connectivity. Together, our findings suggest that higher resilience against the development of cognitive impairment throughout the early stages of Alzheimer's disease is at least partially attributable to higher left frontal cortex-hub connectivity

Transfer of host-derived α synuclein to grafted dopaminergic neurons in rat.

Multiple laboratories have recently demonstrated that long-term dopaminergic transplants form Lewy bodies in patients with Parkinson's disease. Debate has arisen as to whether these Lewy bodies form from the transfer of α synuclein from the host to the graft or whether they form from intrinsic responses of the graft from being placed into what was, or became, an inflammatory focus. To test whether the former hypothesis was possible, we grafted fetal rat ventral mesencephalon into the dopamine depleted striatum of rats that had previously received 6-hydroxydopamine lesions. One month after the transplant, rats received viral over expression of human α synuclein (AAV2/6-α synuclein) or green fluorescent protein (AAV2/6-GFP) into the striatum rostral to the grafts. Care was taken to make sure that the AAV injections were sufficiently distal to the graft so no cells would be directly transfected. All rats were sacrificed five weeks after the virus injections. Double label immunohistochemistry combined with confocal microscopy revealed that a small number of grafted tyrosine hydroxylase (TH) neurons (5.7% ± 1.5% (mean ± SEM) of grafted dopamine cells) expressed host derived α synuclein but none of the grafted cells expressed host-derived GFP. The α synuclein in a few of these cells was misfolded and failed to be digested with proteinase K. These data indicate that it is possible for host derived α synuclein to transfer to grafted neurons supporting the concept that this is one possible mechanism by which grafted dopamine neurons form Lewy bodies in Parkinson's disease patients

Impact of age and vector construct on striatal and nigral transgene expression

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson's disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD

Overexpression of human A53T α-syn impaired mitochondrial respiration and the maximum respiratory capacity.

<p>Bioenergetic parameters of A53T and wt neurons were analyzed using a Seahorse XF96 analyzer as described in Materials and Methods. <b>A</b>, Basal OCR values were similar in wt and A53T neurons. <b>B</b>, A53T neurons had a lower OCR/ECAR ratio than wt both at 7DIV and at 13-14 DIV. <b>C</b>-<b>E</b>, A53T neurons had lower mitochondrial respiration, lower ATP linked respiration, higher non-mitochondrial respiration and lower maximum respiratory capacity than wt neurons at 7DIV (<b>C</b>,<b>D</b>) and at 13-14 DIV (<b>E</b>). Representative data from one experiment for neurons at 7DIV are shown as in C. Pooled data from 3 independent experiments for neurons at 7DIV are shown in D. As in <b>E</b>, pooled data from 3 independent experiments for neurons at 13-14 DIV are shown. Data plotted as Mean +/- SEM (t-test, *p<0.05; **p<0.01; ***p<0.001; ns=non-significant). wt=wild type, A53T=A53T α-synuclein, pMol=picomole, min=minutes, DIV=days <i>in </i><i>vitro</i>, OCR=oxygen consumption rates, ECAR=extracellular acidification rates.</p

Rapamycin rescued mitochondrial mobility in A53T α-syn neurons.

<p><b>A</b>, Representative kymographs showing treatment of rapamycin (1μM) for 7-9hr increased mobility of mitochondria in A53T α-syn neurons (7DIV). Images were acquired at the interval of 12s for 20min. B, C, Rapamycin rescued mitochondrial transport in A53T neurons to the level seen in wild-type cultures. One representative experiment is shown and 58-130 mitochondria were tracked in each condition. Experiments were repeated 3 times and results were consistent across all experiments. <b>D</b>, Rapamycin also rescued mitochondrial length in A53T neurons (6-7DIV) to the level seen in wild-type cultures. The results were pooled from three experiments. Mean +/- SEM is shown and t test was used to compare velocity in different conditions. *p<0.05; **p<0.01; ***p<0.001; ns=non-significant. The error bars depict SEM. wt=wild type, A53T=A53T α-synuclein, μm=micrometers, s=seconds, min=minutes.</p

Transport velocity of autophagosomes was not substantially reduced in A53T α-syn neurons.

<p><b>A</b>, An A53T α-syn cortical neuron transfected with EGFP-LC3 (green) showing diffuse signal in the cytosol and puncta which are autophagosomes. Autophagosomes were visible at the soma (white arrowhead) and a neurite (white and red arrows). <b>B</b>, Montage of images (~1min) from a 5min confocal time-lapse of the neurite area (outlined by red box in A). Two autophagosomes (white and red arrows) moved retrogradely along the neurite. <b>C</b>,<b>D</b>, Representative kymographs of autophagosome mobility demonstrated that most of autophagosomes in wt and A53T neurons underwent active retrograde movement, however, one of them in the wt group (white arrowhead) paused at the end of the imaging period and another one (red arrows) moved anterogradely. <b>E</b>, Bar graphs demonstrating that the average (Avg.) velocity and the instantaneous (Ins.) velocity of autophagosome movement were not reduced in A53T neurons, however, they almost doubled in the mutant cortical neurons. Data were pooled from 3 independent experiments and 309 autophagosomes were included in the analysis. Data plotted as Mean +/- SEM. t test, **p<0.01; ***p<0.001. Scale bar=20μm. wt=wild type, A53T=A53T α-synuclein, μm=micrometers, s=seconds, min=minutes.</p

Mitochondrial transport was reduced in A53T α-syn axons.

<p><b>A</b>, An example of a cortical neuron from A53T mice transfected with dsRed-mito (red) and EGFP (green) at the time of plating. <b>B</b>, Montage of images (~7min) at the dsRed-mito channel from a 20min confocal time-lapse of the neurite area (outlined by white box in A). In the image area, the majority of mitochondria were stationary indicated by the blue and yellow arrows. Red arrows pointed to a mobile mitochondrion that entered the area at ~200s and moved along the axon. <b>C</b>, <b>D</b>, Kymographs constructed from five-pixel wide lines drawn through the extent of axons from wt or A53T neurons. Fluorescence intensities along kymograph lines are plotted horizontally for each frame of the 5min time-lapse with time interval of 3s (101 frames). Time is indicated on the y-axis and distance is indicated on the x-axis. In A53T neurons (<b>C</b>), most of mitochondria are stationary represented by the bright vertical lines. In wt neurons (<b>D</b>), there are diagonal lines representing moving mitochondria. <b>E</b>, <b>F</b>, Bar graphs from one representative experiment at 7DIV (<b>E</b>) and 14DIV (<b>F</b>) showing that mitochondria transport velocity was reduced significantly in A53T neurons. In each condition, 53-145 mitochondria were analyzed and ≥ 4 independent experiments showed consistent results. <b>G</b>, Percentage of mobile mitochondria significantly decreased in A53T (~8%) compared to that in wt neurons (>30%). Mobile mitochondria are defined as mitochondria that moved at ≥0.005 μm/s. H, Mitochondrial instantaneous velocity also reduced in A53T axons. Data plotted as Mean +/- SEM. t test, *p<0.05; **p<0.01; ****p<0.0001. Scale bar=20μm. wt=wild type, A53T=A53T α-synuclein, μm=micrometers, s=seconds, min=minutes, DIV=days <i>in </i><i>vitro</i>.</p

Human A53T α-Synuclein Causes Reversible Deficits in Mitochondrial Function and Dynamics in Primary Mouse Cortical Neurons

<div><p>Parkinson’s disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is α-synuclein (α-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the α-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant α-syn gene develop severe movement disorders. However, the molecular mechanisms of α-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson’s disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T α-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.</p> </div