Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP ßCTF.

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 ß-C-terminal fragment (ßCTF) to disrupt lysosomal vacuolar (H+)-adenosine triphosphatase (v-ATPase) and acidification. In human DS fibroblasts, the phosphorylated 682YENPTY internalization motif of APP-ßCTF 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-ßCTF Tyr682 phosphorylation restores v-ATPase and lysosome function in DS fibroblasts and in vivo in brains of DS model mice. Notably, lowering APP-ßCTF Tyr682 phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-ßCTF. Elevated APP-ßCTF Tyr682 phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer previously unknown insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.

Autophagy is a novel pathway for neurofilament protein degradation in vivo.

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.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration.

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.

Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer's disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-ß secretase cleaved C-terminal fragment (APP-ßCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-ßCTF 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-3ß. 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 ß-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.

Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice.

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

Marked calpastatin (CAST) depletion in Alzheimer's disease accelerates cytoskeleton disruption and neurodegeneration: neuroprotection by CAST overexpression.

Increased activity of calpains is implicated in synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). The molecular mechanisms responsible for increased calpain activity in AD are not known. Here, we demonstrate that disease progression is propelled by a marked depletion of the endogenous calpain inhibitor, calpastatin (CAST), from AD neurons, which is mediated by caspase-1, caspase-3, and calpains. Initial CAST depletion focally along dendrites coincides topographically with calpain II and ERK 1/2 activation, tau cleavage by caspase-3, and tau and neurofilament hyperphosphorylation. These same changes, together with cytoskeletal proteolysis and neuronal cell death, accompany CAST depletion after intrahippocampal kainic acid administration to mice, and are substantially reduced in mice overexpressing human CAST. Moreover, CAST reduction by shRNA in neuronal cells causes calpain-mediated death at levels of calcium-induced injury that are sublethal to cells normally expressing CAST. Our results strongly support a novel hypothesis that CAST depletion by multiple abnormally activated proteases accelerates calpain dysregulation in AD leading to cytoskeleton disruption and neurodegeneration. CAST mimetics may, therefore, be neuroprotective in AD.