Alzheimer's disease: insights into pathology, molecular mechanisms, and therapy.

Alzheimer's disease (AD), the leading cause of dementia, is characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. This condition casts a significant shadow on global health due to its complex and multifactorial nature. In addition to genetic predispositions, the development of AD is influenced by a myriad of risk factors, including aging, systemic inflammation, chronic health conditions, lifestyle and environmental exposures. Recent advancements in understanding the complex pathophysiology of AD are paving the way for enhanced diagnostic techniques, improved risk assessment, and potentially effective prevention strategies. These discoveries are crucial in the quest to unravel the complexities of AD, offering a beacon of hope for improved management and treatment options for the millions affected by this debilitating disease.

ß2-Microglobulin coaggregates with Aß and contributes to amyloid pathology and cognitive deficits in Alzheimer's disease model mice.

Extensive studies indicate that ß-amyloid (Aß) aggregation is pivotal for Alzheimer's disease (AD) progression; however, cumulative evidence suggests that Aß itself is not sufficient to trigger AD-associated degeneration, and whether other additional pathological factors drive AD pathogenesis remains unclear. Here, we characterize pathogenic aggregates composed of ß2-microglobulin (ß2M) and Aß that trigger neurodegeneration in AD. ß2M, a component of major histocompatibility complex class I (MHC class I), is upregulated in the brains of individuals with AD and constitutes the amyloid plaque core. Elevation of ß2M aggravates amyloid pathology independent of MHC class I, and coaggregation with ß2M is essential for Aß neurotoxicity. B2m genetic ablation abrogates amyloid spreading and cognitive deficits in AD mice. Antisense oligonucleotide- or monoclonal antibody-mediated ß2M depletion mitigates AD-associated neuropathology, and inhibition of ß2M-Aß coaggregation with a ß2M-based blocking peptide ameliorates amyloid pathology and cognitive deficits in AD mice. Our findings identify ß2M as an essential factor for Aß neurotoxicity and a potential target for treating AD.

ß2-microglobulin functions as an endogenous NMDAR antagonist to impair synaptic function.

Down syndrome (DS) is a neurological disorder with multiple immune-related symptoms; however, crosstalk between the CNS and peripheral immune system remains unexplored. Using parabiosis and plasma infusion, we found that blood-borne factors drive synaptic deficits in DS. Proteomic analysis revealed elevation of ß2-microglobulin (B2M), a major histocompatibility complex class I (MHC-I) component, in human DS plasma. Systemic administration of B2M in wild-type mice led to synaptic and memory defects similar to those observed in DS mice. Moreover, genetic ablation of B2m or systemic administration of an anti-B2M antibody counteracts synaptic impairments in DS mice. Mechanistically, we demonstrate that B2M antagonizes NMDA receptor (NMDAR) function through interactions with the GluN1-S2 loop; blocking B2M-NMDAR interactions using competitive peptides restores NMDAR-dependent synaptic function. Our findings identify B2M as an endogenous NMDAR antagonist and reveal a pathophysiological role for circulating B2M in NMDAR dysfunction in DS and related cognitive disorders.

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice.

Synaptic abnormality is an important pathologic feature of autism spectrum disorders (ASDs) and responsible for various behavioral defects in these neurodevelopmental disorders. Microglia are the major immune cells in the brain and also play an important role in synapse refinement. Although dysregulated synaptic pruning by microglia during the brain development has been associated with ASDs, the underlying mechanism has yet to be fully elucidated. Herein, we observed that expression of Transmembrane protein 59 (TMEM59), a protein recently shown to regulate microglial function, was decreased in autistic patients. Furthermore, we found that both male and female mice with either complete or microglia-specific loss of Tmem59 developed ASD-like behaviors. Microglial TMEM59-deficient mice also exhibited enhanced excitatory synaptic transmission, increased dendritic spine density, and elevated levels of excitatory synaptic proteins in synaptosomes. TMEM59-deficient microglia had impaired capacity for synapse engulfment both in vivo and in vitro. Moreover, we demonstrated that TMEM59 interacted with the C1q receptor CD93 and TMEM59 deficiency promoted CD93 protein degradation in microglia. Downregulation of CD93 in microglia also impaired synapse engulfment. These findings identify a crucial role of TMEM59 in modulating microglial function on synapse refinement during brain development and suggest that TMEM59 deficiency may contribute to ASDs through disrupting phagocytosis of excitatory synapse and thus distorting the excitatory-inhibitory (E/I) neuronal activity balance.SIGNIFICANCE STATEMENT Microglia play an important role in synapse refinement. Dysregulated synaptic pruning by microglia during brain development has been associated with autism spectrum disorders (ASDs). However, the underlying mechanism has yet to be fully elucidated. Herein, we observe that the expression of Transmembrane protein 59 (TMEM59), an autophagy-related protein, is decreased in autistic patients. Moreover, we find ASD-like behaviors in mice with complete loss and with microglia-specific loss of Tmem59 Mechanistic studies reveal that TMEM59 deficiency in microglia impairs their synapse engulfment ability likely through destabilizing the C1q receptor CD93, thereby leading to enhanced excitatory neurotransmission and increased dendritic spine density. Our findings demonstrate a crucial role of microglial TMEM59 in early neuronal development and provide new insight into the etiology of ASDs.

Metabolic reprogramming in astrocytes results in neuronal dysfunction in intellectual disability.

Astrocyte aerobic glycolysis provides vital trophic support for central nervous system neurons. However, whether and how astrocytic metabolic dysregulation contributes to neuronal dysfunction in intellectual disability (ID) remain unclear. Here, we demonstrate a causal role for an ID-associated SNX27 mutation (R198W) in cognitive deficits involving reshaping astrocytic metabolism. We generated SNX27R196W (equivalent to human R198W) knock-in mice and found that they displayed deficits in synaptic function and learning behaviors. SNX27R196W resulted in attenuated astrocytic glucose uptake via GLUT1, leading to reduced lactate production and a switch from homeostatic to reactive astrocytes. Importantly, lactate supplementation or a ketogenic diet restored neuronal oxidative phosphorylation and reversed cognitive deficits in SNX27R196W mice. In summary, we illustrate a key role for astrocytic SNX27 in maintaining glucose supply and glycolysis and reveal that altered astrocytic metabolism disrupts the astrocyte-neuron interaction, which contributes to ID. Our work also suggests a feasible strategy for treating ID by restoring astrocytic metabolic function.

USP25 inhibition ameliorates Alzheimer's pathology through the regulation of APP processing and Aß generation.

Down syndrome (DS), or trisomy 21, is one of the critical risk factors for early-onset Alzheimer's disease (AD), implicating key roles for chromosome 21-encoded genes in the pathogenesis of AD. We previously identified a role for the deubiquitinase USP25, encoded on chromosome 21, in regulating microglial homeostasis in the AD brain; however, whether USP25 affects amyloid pathology remains unknown. Here, by crossing 5×FAD AD and Dp16 DS mice, we observed that trisomy 21 exacerbated amyloid pathology in the 5×FAD brain. Moreover, bacterial artificial chromosome (BAC) transgene-mediated USP25 overexpression increased amyloid deposition in the 5×FAD mouse brain, whereas genetic deletion of Usp25 reduced amyloid deposition. Furthermore, our results demonstrate that USP25 promoted ß cleavage of APP and Aß generation by reducing the ubiquitination and lysosomal degradation of both APP and BACE1. Importantly, pharmacological inhibition of USP25 ameliorated amyloid pathology in the 5×FAD mouse brain. In summary, we identified the DS-related gene USP25 as a critical regulator of AD pathology, and our data suggest that USP25 serves as a potential pharmacological target for AD drug development.

SNX14 deficiency-induced defective axonal mitochondrial transport in Purkinje cells underlies cerebellar ataxia and can be reversed by valproate.

Loss-of-function mutations in sorting nexin 14 (SNX14) cause autosomal recessive spinocerebellar ataxia 20, which is a form of early-onset cerebellar ataxia that lacks molecular mechanisms and mouse models. We generated Snx14-deficient mouse models and observed severe motor deficits and cell-autonomous Purkinje cell degeneration. SNX14 deficiency disrupted microtubule organization and mitochondrial transport in axons by destabilizing the microtubule-severing enzyme spastin, which is implicated in dominant hereditary spastic paraplegia with cerebellar ataxia, and compromised axonal integrity and mitochondrial function. Axonal transport disruption and mitochondrial dysfunction further led to degeneration of high-energy-demanding Purkinje cells, which resulted in the pathogenesis of cerebellar ataxia. The antiepileptic drug valproate ameliorated motor deficits and cerebellar degeneration in Snx14-deficient mice via the restoration of mitochondrial transport and function in Purkinje cells. Our study revealed an unprecedented role for SNX14-dependent axonal transport in cerebellar ataxia, demonstrated the convergence of SNX14 and spastin in mitochondrial dysfunction, and suggested valproate as a potential therapeutic agent.

Trisomy 21-induced dysregulation of microglial homeostasis in Alzheimer's brains is mediated by USP25.

Down syndrome (DS), caused by trisomy of chromosome 21, is the most significant risk factor for early-onset Alzheimer's disease (AD); however, underlying mechanisms linking DS and AD remain unclear. Here, we show that triplication of homologous chromosome 21 genes aggravates neuroinflammation in combined murine DS-AD models. Overexpression of USP25, a deubiquitinating enzyme encoded by chromosome 21, results in microglial activation and induces synaptic and cognitive deficits, whereas genetic ablation of Usp25 reduces neuroinflammation and rescues synaptic and cognitive function in 5×FAD mice. Mechanistically, USP25 deficiency attenuates microglia-mediated proinflammatory cytokine overproduction and synapse elimination. Inhibition of USP25 reestablishes homeostatic microglial signatures and restores synaptic and cognitive function in 5×FAD mice. In summary, we demonstrate an unprecedented role for trisomy 21 and pathogenic effects associated with microgliosis as a result of the increased USP25 dosage, implicating USP25 as a therapeutic target for neuroinflammation in DS and AD.

Overexpression of Human SNX27 Enhances Learning and Memory Through Modulating Synaptic Plasticity in Mice.

Abnormal synaptic transmission leads to learning and memory disorders and is the main feature of neurological diseases. Sorting nexin 27 (SNX27) is an endosomal adaptor protein associated with a variety of nervous system diseases, and it is mainly responsible for the trafficking of postsynaptic membrane receptors. However, the roles of SNX27 in regulating synaptic and cognitive function are not fully understood. Here, we first generated a neuron-specific human-SNX27 transgenic mouse model (hSNX27 Tg) that exhibited enhanced excitatory synaptic transmission and long-term potentiation (LTP). In addition, we found that the hSNX27 Tg mice displayed enhanced learning and memory, lower-level anxiety-like behavior, and increased social interaction. Furthermore, we found that SNX27 overexpression upregulated the expression of glutamate receptors in the cortex and hippocampus of hSNX27 Tg mice. Together, these results indicate that SNX27 overexpression promotes synaptic function and cognition through modulating glutamate receptors.

The deubiquitinase USP6 affects memory and synaptic plasticity through modulating NMDA receptor stability.

Ubiquitin-specific protease (USP) 6 is a hominoid deubiquitinating enzyme previously implicated in intellectual disability and autism spectrum disorder. Although these findings link USP6 to higher brain function, potential roles for USP6 in cognition have not been investigated. Here, we report that USP6 is highly expressed in induced human neurons and that neuron-specific expression of USP6 enhances learning and memory in a transgenic mouse model. Similarly, USP6 expression regulates N-methyl-D-aspartate-type glutamate receptor (NMDAR)-dependent long-term potentiation and long-term depression in USP6 transgenic mouse hippocampi. Proteomic characterization of transgenic USP6 mouse cortex reveals attenuated NMDAR ubiquitination, with concomitant elevation in NMDAR expression, stability, and cell surface distribution with USP6 overexpression. USP6 positively modulates GluN1 expression in transfected cells, and USP6 down-regulation impedes focal GluN1 distribution at postsynaptic densities and impairs synaptic function in neurons derived from human embryonic stem cells. Together, these results indicate that USP6 enhances NMDAR stability to promote synaptic function and cognition.

Inhibition of PKCδ reduces amyloid-ß levels and reverses Alzheimer disease phenotypes.

ß-amyloid protein (Aß) plays a central role in the pathogenesis of Alzheimer disease (AD). Aß is generated from sequential cleavage of amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. Although activation of some protein kinase C (PKC) isoforms such as PKCα and ε has been shown to regulate nonamyloidogenic pathways and Aß degradation, it is unclear whether other PKC isoforms are involved in APP processing/AD pathogenesis. In this study, we report that increased PKCδ levels correlate with BACE1 expression in the AD brain. PKCδ knockdown reduces BACE1 expression, BACE1-mediated APP processing, and Aß production. Conversely, overexpression of PKCδ increases BACE1 expression and Aß generation. Importantly, inhibition of PKCδ by rottlerin markedly reduces BACE1 expression, Aß levels, and neuritic plaque formation and rescues cognitive deficits in an APP Swedish mutations K594N/M595L/presenilin-1 with an exon 9 deletion-transgenic AD mouse model. Our study indicates that PKCδ plays an important role in aggravating AD pathogenesis, and PKCδ may be a potential target in AD therapeutics.

Snx27 Deletion Promotes Recovery From Spinal Cord Injury by Neuroprotection and Reduces Macrophage/Microglia Proliferation.

Sorting nexin 27 (SNX27) is an endosome-associated cargo adaptor that is involved in various pathologies and development of neurological diseases. However, the role of SNX27 in spinal cord injury (SCI) remains unclear. In this study, we found that SNX27 was up-regulated in injured mice spinal cords by western blot and immunofluorescence. A comparative analysis of Basso mouse scale (BMS), footprint test and corticospinal tract (CST) tracing in Snx27 +/+ and Snx27 +/- mice revealed that haploinsufficiency of SNX27 ameliorated the clinical symptoms of SCI. Based on the results of western blot and immunofluorescence, mechanistically, we found that SNX27 deficiency suppresses apoptotic caspase-3 induced neuronal death. In addition, SNX27 haploinsufficiency lowers the infiltration and activation of macrophage/microglia by suppressing their proliferation at the SCI lesion site. Together, these results suggest that down-regulation of SNX27 is a potential therapy targeting both acute neuronal death and chronic neuroinflammation, and promoting nerve repair after SCI.

The Neuron-Specific Protein TMEM59L Mediates Oxidative Stress-Induced Cell Death.

TMEM59L is a newly identified brain-specific membrane-anchored protein with unknown functions. Herein we found that both TMEM59L and its homolog, TMEM59, are localized in Golgi and endosomes. However, in contrast to a ubiquitous and relatively stable temporal expression of TMEM59, TMEM59L expression was limited in neurons and increased during development. We also found that both TMEM59L and TMEM59 interacted with ATG5 and ATG16L1, and that overexpression of them triggered cell autophagy. However, overexpression of TMEM59L induced intrinsic caspase-dependent apoptosis more dramatically than TMEM59. In addition, downregulation of TMEM59L prevented neuronal cell death and caspase-3 activation caused by hydrogen peroxide insults and reduced the lipidation of LC3B. Finally, we found that AAV-mediated knockdown of TMEM59L in mice significantly ameliorated caspase-3 activation, increased mouse duration in the open arm during elevated plus maze test, reduced mouse immobility time during forced swim test, and enhanced mouse memory during Y-maze and Morris water maze tests. Together, our study indicates that TMEM59L is a pro-apoptotic neuronal protein involved in animal behaviors such as anxiety, depression, and memory, and that TMEM59L downregulation protects neurons against oxidative stress.

Dysregulation of Ubiquitin-Proteasome System in Neurodegenerative Diseases.

The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways, where abnormal UPS function has been observed in cancer and neurological diseases. Many neurodegenerative diseases share a common pathological feature, namely intracellular ubiquitin-positive inclusions formed by aggregate-prone neurotoxic proteins. This suggests that dysfunction of the UPS in neurodegenerative diseases contributes to the accumulation of neurotoxic proteins and to instigate neurodegeneration. Here, we review recent findings describing various aspects of UPS dysregulation in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis.

Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. SIGNIFICANCE STATEMENT: Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of therapeutic interest to congenital hydrocephalus.

Appoptosin-Mediated Caspase Cleavage of Tau Contributes to Progressive Supranuclear Palsy Pathogenesis.

Progressive supranuclear palsy (PSP) is a movement disorder characterized by tau neuropathology where the underlying mechanism is unknown. An SNP (rs1768208 C/T) has been identified as a strong risk factor for PSP. Here, we identified a much higher T-allele occurrence and increased levels of the pro-apoptotic protein appoptosin in PSP patients. Elevations in appoptosin correlate with activated caspase-3 and caspase-cleaved tau levels. Appoptosin overexpression increased caspase-mediated tau cleavage, tau aggregation, and synaptic dysfunction, whereas appoptosin deficiency reduced tau cleavage and aggregation. Appoptosin transduction impaired multiple motor functions and exacerbated neuropathology in tau-transgenic mice in a manner dependent on caspase-3 and tau. Increased appoptosin and caspase-3-cleaved tau were also observed in brain samples of patients with Alzheimer's disease and frontotemporal dementia with tau inclusions. Our findings reveal a novel role for appoptosin in neurological disorders with tau neuropathology, linking caspase-3-mediated tau cleavage to synaptic dysfunction and behavioral/motor defects.

Role of copper and the copper-related protein CUTA in mediating APP processing and Aß generation.

One major pathologic hallmark and trigger of Alzheimer's disease (AD) is overproduction and accumulation of ß-amyloid (Aß) species in the brain. Aß is derived from ß-amyloid precursor protein (APP) through sequential cleavages by ß- and γ-secretases. Abnormal copper homeostasis also contributes to AD pathogenesis. Recently, we find that a copper-related protein, CutA divalent cation tolerance homolog of Escherichia coli (CUTA), interacts with the ß-secretase ß-site APP cleaving enzyme 1 (BACE1) and inhibits APP ß-processing and Aß generation. Herein, we further found that overexpression of CUTA increases intracellular copper level, whereas copper treatments promote CUTA expression. We also confirmed that copper treatments promote APP expression and Aß secretion. In addition, copper treatments promoted the increase of Aß secretion induced by CUTA downregulation but had no effect on CUTA-ß-site APP cleaving enzyme 1 interaction. On the other hand, CUTA overexpression ameliorated copper-induced Aß secretion but had no effect on APP expression. Moreover, we found that Aß treatments can reduce both CUTA and copper levels in mouse primary neurons. Consistently, both CUTA and copper levels were decreased in the hippocampus of APP/PS1 AD mouse brain. Together, our results reveal a reciprocal modulation of copper and CUTA and suggest that both regulate Aß generation through different mechanisms, although Aß mutually affects copper and CUTA levels.

Sorting nexin 27 regulates Aß production through modulating γ-secretase activity.

Patients with Down syndrome (DS) invariably develop Alzheimer's disease (AD) pathology in their 40s. We have recently found that overexpression of a chromosome 21-encoded microRNA-155 results in decreased levels of the membrane trafficking component, SNX27, diminishing glutamate receptor recycling and thereby impairing synaptic functions in DS. Here, we report a function of SNX27 in regulating ß-amyloid (Aß) generation by modulating γ-secretase activity. Downregulation of SNX27 using RNAi increased Aß production, whereas overexpression of full-length SNX27, but not SNX27ΔPDZ, reversed the RNAi-mediated Aß elevation. Moreover, genetic deletion of Snx27 promoted Aß production and neuronal loss, whereas overexpression of SNX27 using an adeno-associated viral (AAV) vector reduced hippocampal Aß levels in a transgenic AD mouse model. SNX27 associates with the γ-secretase complex subunit presenilin 1; this interaction dissociates the γ-secretase complex, thus decreasing its proteolytic activity. Our study establishes a molecular mechanism for Aß-dependent pathogenesis in both DS and AD.

Ferritin light chain interacts with PEN-2 and affects γ-secretase activity.

Alzheimer's disease (AD) is primarily caused by overproduction/deposition of ß-amyloid (Aß) in the brain. Dysregulation of iron in the brain also contributes to AD. Although iron affects ß-amyloid precursor protein (APP) expression and Aß deposition, detailed role of iron in AD requires further elucidation. Aß is produced by sequential proteolytic cleavages of APP by ß-secretase and γ-secretase. The γ-secretase complex comprises presenilins (PS1 or PS2), nicastrin, APH-1, and PEN-2. Herein, we find that PEN-2 can interact with ferritin light chain (FTL), an important component of the iron storage protein ferritin. In addition, we show that overexpression of FTL increases the protein levels of PEN-2 and PS1 amino-terminal fragment (NTF) and promotes γ-secretase activity for more production of Aß and notch intracellular domain (NICD). Furthermore, iron treatments increase the levels of FTL, PEN-2 and PS1 NTF and promote γ-secretase-mediated NICD production. Moreover, downregulation of FTL decreases the levels of PEN-2 and PS1 NTF. Together, our results suggest that iron can increase γ-secretase activity through promoting the level of FTL that interacts with and stabilizes PEN-2, providing a new molecular link between iron, PEN-2/γ-secretase and Aß generation in AD.