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 ␤ cleaved carboxy terminal fragment of APP (APP-␤CTF, 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-␤CTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-␤CTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD.

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.

Highlights d Pathological Rab5 activation in PA-Rab5 mice mimics AD-like endosomal dysfunction d PA-Rab5 mice have synaptic function/structure deficits and GSK-3b-mediated tauopathy d Rab5 overactivation in vivo underlies cholinergic degeneration and memory deficits d Endosomal dysfunction alone induces prodromal and degenerative AD-related changes

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.

We previously characterized a H+ transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by NAD(P)H oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13BN) 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. In order 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 NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1−/− rats (P<0.05), and more rapid recovery of intracellular pH in wild-type rats (ΔpHi 0.005U/sec vs. 0.002U/sec, p=0.046 respectively). Superoxide production was enhanced by low intracellular sodium (<10mM) in both thick limb and peritoneal macrophages only when HV1 was present. When fed a high salt diet, blood pressure, outer-medullary renal injury (tubular casts) and oxidative stress (4-Hydroxynonenal staining) were significantly reduced in HV1−/− rats compared to 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.

The endosome-associated GTPase 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 is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

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