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How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives

Lysosome dysfunction arises early and propels Alzheimer’s Disease (AD). Herein, we show that amyloid precursor protein (APP), linked to early-onset AD in Down Syndrome (DS), acts directly via its β-C-terminal fragment (βCTF) to disrupt lysosomal v-ATPase and acidification. In human DS fibroblasts, the phosphorylated682YENPTY internalization motif of APP-βCTF binds selectively within a pocket of the v-ATPase V0a1 subunit cytoplasmic domain and competitively inhibits association of the V1 subcomplex of v-ATPase, thereby reducing its activity. Lowering APP-βCTF Tyr682phosphorylation restores v-ATPase and lysosome function in DS fibroblasts andin vivoin brains of DS model mice. Notably, lowering APP-βCTF Tyr682phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-βCTF. Elevated APP-βCTF Tyr682phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer new insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.

Summary Lysosomal dysfunction is considered pathogenic in Alzheimer Disease (AD). Loss of Presenilin-1 (PSEN1) function causing AD impedes acidification via defective vATPase V0a1 subunit delivery to lysosomes. We report that isoproterenol and related β2-adrenergic agonists re-acidify lysosomes in PSEN1 KO cells and fibroblasts from PSEN1 familial AD(FAD) patients, which restores lysosomal proteolysis, calcium homeostasis, and normal autophagy flux. We identify a novel rescue mechanism involving PKA-mediated facilitation of ClC-7 delivery to lysosomes which reverses markedly lowered Cl- content in PSEN1 KO lysosomes. Notably, PSEN1 loss-of-function impedes ER-to-lysosome delivery of ClC-7. Transcriptomics of PSEN1-deficient cells reveal strongly down-regulated ER-to-lysosome transport pathways and reversibility by isoproterenol thus accounting for lysosomal Cl- deficits that compound pH elevation due to deficient vATPase and rescue by β2-adrenergic agonists. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β2-adrenergic regulation of ClC-7, which can potentially be modulated therapeutically.

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.

The endosome-associated protein Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α kinase is a Rab-5 activator, we hypothesized that inhibition of this kinase held potential as an approach to treat diseases associated with BFCN loss. Herein we report that treatment with an oral small molecule p38α kinase inhibitor reversed pathological disease progression in the basal forebrain in a mouse model that develops BFCN degeneration. Further, the preclinical results were successfully translated to the clinic, with improvement of clinical outcomes associated with cholinergic function in a clinical study in dementia with Lewy bodies (DLB), a disease in which BFCN dysfunction and degeneration is the primary driver of disease expression. The findings both advances a novel approach to treating DLB and validates the translational platform that provided the mechanistic rationale for advancing that approach.

Neurofilament (NFL) proteins have recently been found to play unique roles in synapses. NFL is known to interact with the GluN1 subunit of N-methyl-d-aspartic acid (NMDAR) and be reduced in schizophrenia though functional consequences are unknown. Here we investigated whether the interaction of NFL with GluN1 modulates synaptic transmission and schizophrenia-associated behaviors. The interaction of NFL with GluN1 was assessed by means of molecular, pharmacological, electrophysiological, magnetic resonance spectroscopy (MRS), and schizophrenia-associated behavior analyses. NFL deficits cause an NMDAR hypofunction phenotype including abnormal hippocampal function, as seen in schizophrenia. NFL−/− deletion in mice reduces dendritic spines and GluN1 protein levels, elevates ubiquitin-dependent turnover of GluN1 and hippocampal glutamate measured by MRS, and depresses hippocampal long-term potentiation. NMDAR-related behaviors are also impaired, including pup retrieval, spatial and social memory, prepulse inhibition, night-time activity, and response to NMDAR antagonist, whereas motor deficits are minimal. Importantly, partially lowering NFL in NFL+/− mice to levels seen regionally in schizophrenia, induced similar but milder NMDAR-related synaptic and behavioral deficits. Our findings support an emerging view that central nervous system neurofilament subunits including NFL in the present report, serve distinctive, critical roles in synapses relevant to neuropsychiatric diseases.

Down syndrome (DS), a complex genetic disorder caused by chromosome 21 trisomy, is associated with mitochondrial dysfunction leading to the accumulation of damaged mitochondria. Here we report that mitophagy, a form of selective autophagy activated to clear damaged mitochondria is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. We identified two molecular bases for this mitophagy deficiency: PINK1/PARKIN impairment and abnormal suppression of macroautophagy. First, strongly downregulated PARKIN and the mitophagic adaptor protein SQSTM1/p62 delays PINK1 activation to impair mitophagy induction after mitochondrial depolarization by CCCP or antimycin A plus oligomycin. Secondly, mTOR is strongly hyper-activated, which globally suppresses macroautophagy induction and the transcriptional expression of proteins critical for autophagosome formation such as ATG7, ATG3 and FOXO1. Notably, inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) using AZD8055 (AZD) restores autophagy flux, PARKIN/PINK initiation of mitophagy, and the clearance of damaged mitochondria by mitophagy. These results recommend mTORC1-mTORC2 inhibition as a promising candidate therapeutic strategy for Down Syndrome. post-print 3199 KB

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the β cleaved carboxy terminal fragment of APP (APP-βCTF, C99). In primary fibroblasts from individuals with DS, lysosomal degradation of autophagic and endocytic substrates is selectively impaired, causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, whereas RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one Bace1 allele from adult Ts2 mice had similar rescue effects in vivo. The modest elevation of endogenous APP-βCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-βCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD. SIGNIFICANCE STATEMENT Down syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer's disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein (APP) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates, processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous β-cleaved carboxy-terminal fragment of APP (APP-βCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-βCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer’s disease (AD), is mediated by aberrant activation of Rab5 triggered by APP-βeta secretase cleaved C-terminal fragment (APP-βCTF). To distinguish pathophysiological consequences specific to over-activated Rab5 itself, we activated Rab5 independently from APP-βCTF in a novel mouse model (PA-Rab5). We report that Rab5 over-activation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyper-activation of Rab5 comparable to that in AD brain reproduced AD-related Rab5-endosomal enlargement and mistrafficking; hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss; and tau hyperphosphorylation via activated glycogen synthase kinase-3β. Importantly, Rab5-mediated endosomal dysfunction induced progressive cholinergic neurodegeneration and impaired hippocampal dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a novel pathogenic cascade distinct from β-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 over-activation as a new therapeutic target.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer’s disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-β secretase cleaved C-terminal fragment (APP-βCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-βCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3β. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from β-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.

We previously characterized a H + transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13 BN ) rats, and superoxide production was enhanced in low Na + media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na + . We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H + currents. To test this hypothesis, we developed HV1 −/− null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH 4 Cl prepulse in 0 sodium/BaCl 2 containing media resulted in superoxide production in thick limb from wild-type but not HV1 −/− rats ( P I 0.005 versus 0.002 U/s, P =0.046, respectively). Superoxide production was enhanced by low intracellular sodium (−/− rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na + is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.

Patients undergoing major lower extremity amputation (MLEA) for peripheral arterial disease are often elderly, debilitated, and fraught with medical comorbid conditions that place them at high risk for surgical intervention. Data from lower extremity revascularization surgeries are often extrapolated to determine which anesthetic modality to use for amputations, with preference given to regional anesthesia. However, there is little evidence to support the use of one mode of anesthesia over another. We conducted this study to determine the effect of anesthetic modality on the clinical outcomes of patients undergoing above- or below-knee amputations.This study is a retrospective review of consecutive patients who underwent MLEA at a single center between 2002-2011. The study population was divided into 2 groups based on anesthetic modality (i.e., regional vs. general anesthesia). These groups were compared based on demographics and comorbidities. Major outcomes analyzed included death, myocardial infarction (MI), and pulmonary complications. Secondary outcome measures included cardiac arrhythmias, venous thromboembolism (VTE), and duration of stay in the intensive care unit and hospital.Four hundred sixty-three patients were identified; 56 patients were excluded for incomplete data, leaving 407 patients in the 2 groups combined. Of these, 259 patients underwent amputation under regional anesthesia; 148 underwent amputation under general anesthesia. Patients in the regional anesthesia group were older (76.6 vs. 71.6 years; P=0.001) and had a lower body mass index (25.2 vs. 26.9 kg/m2; P=0.013). They were also less likely to be on preoperative antiplatelet therapy (aspirin or clopidogrel) or anticoagulation (27% vs. 45%; P0.001). Regional anesthesia was associated with a lower incidence of overall postoperative pulmonary complications (15% vs. 24%; P=0.02) and postoperative arrhythmia (14% vs. 25%; P=0.001). Duration of stay in the intensive care unit (1.92 vs. 3.85 days; P=0.001) and hospital (19.4 vs 23.1 days; P=0.037) were significantly longer in the group receiving general anesthesia. No significant differences in postoperative MI (12% vs. 9%; P=not significant [NS]), VTE (5% vs. 7%; P=NS) or mortality (10% vs. 13%; P=NS) was seen between groups. Controlling for procedure, above- versus below-knee amputation did not significantly alter these results.Regional anesthesia for patients undergoing MLEA is associated with a lower incidence of postoperative pulmonary complications and cardiac arrhythmias. It is also associated with lower resource use. As such, regional anesthesia should likely be the favored anesthetic modality for patients undergoing MLEA.