Diminished Neuronal ESCRT-0 Function Exacerbates AMPA Receptor Derangement and Accelerates Prion-Induced Neurodegeneration.

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrPC, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrPC), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrPC-mediated neurotoxic signaling.

BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

Novel systemic delivery of a peptide-conjugated antisense oligonucleotide to reduce α-synuclein in a mouse model of Alzheimer's disease.

Neurodegenerative disorders of aging are characterized by the progressive accumulation of proteins such as α-synuclein (α-syn) and amyloid beta (Aß). Misfolded and aggregated α-syn has been implicated in neurological disorders such as Parkinson's disease, and Dementia with Lewy Bodies, but less so in Alzheimer's Disease (AD), despite the fact that accumulation of α-syn has been confirmed in over 50% of postmortem brains neuropathologically diagnosed with AD. To date, no therapeutic strategy has effectively or consistently downregulated α-syn in AD. Here we tested the hypothesis that by using a systemically-delivered peptide (ApoB11) bound to a modified antisense oligonucleotide against α-syn (ASO-α-syn), we can downregulate α-syn expression in an AD mouse model and improve behavioral and neuropathologic phenotypes. Our results demonstrate that monthly systemic treatment with of ApoB11:ASO α-syn beginning at 6 months of age reduces expression of α-synuclein in the brains of 9-month-old AD mice. Downregulation of α-syn led to reduction in Aß plaque burden, prevented neuronal loss and astrogliosis. Furthermore, we found that AD mice treated with ApoB11:ASO α-syn had greatly improved hippocampal and spatial memory function in comparison to their control counterparts. Collectively, our data supports the reduction of α-syn through use of systemically-delivered ApoB11:ASO α-syn as a promising future disease-modifying therapeutic for AD.

Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy.

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimer's disease (AD) in PD patients.

Prions induce an early Arc response and a subsequent reduction in mGluR5 in the hippocampus.

Synapse dysfunction and loss are central features of neurodegenerative diseases, caused in part by the accumulation of protein oligomers. Amyloid-ß, tau, prion, and α-synuclein oligomers bind to the cellular prion protein (PrPC), resulting in the activation of macromolecular complexes and signaling at the post-synapse, yet the early signaling events are unclear. Here we sought to determine the early transcript and protein alterations in the hippocampus during the pre-clinical stages of prion disease. We used a transcriptomic approach focused on the early-stage, prion-infected hippocampus of male wild-type mice, and identify immediate early genes, including the synaptic activity response gene, Arc/Arg3.1, as significantly upregulated. In a longitudinal study of male, prion-infected mice, Arc/Arg-3.1 protein was increased early (40% of the incubation period), and by mid-disease (pre-clinical), phosphorylated AMPA receptors (pGluA1-S845) were increased and metabotropic glutamate receptors (mGluR5 dimers) were markedly reduced in the hippocampus. Notably, sporadic Creutzfeldt-Jakob disease (sCJD) post-mortem cortical samples also showed low levels of mGluR5 dimers. Together, these findings suggest that prions trigger an early Arc response, followed by an increase in phosphorylated GluA1 and a reduction in mGluR5 receptors.

RAGE: A potential therapeutic target during FGF1 treatment of diabetes-mediated liver injury.

As a serious metabolic disease, diabetes causes series of complications that seriously endanger human health. The liver is a key organ for metabolizing glucose and lipids, which substantially contributes to the development of insulin resistance and type 2 diabetes mellitus (T2DM). Exogenous fibroblast growth factor 1 (FGF1) has a great potential for the treatment of diabetes. Receptor of advanced glycation end products (RAGE) is a receptor for advanced glycation end products that involved in the development of diabetes-triggered complications. Previous study has demonstrated that FGF1 significantly ameliorates diabetes-mediated liver damage (DMLD). However, whether RAGE is involved in this process is still unknown. In this study, we intraperitoneally injected db/db mice with 0.5 mg/kg FGF1. We confirmed that FGF1 treatment not only significantly ameliorates diabetes-induced elevated apoptosis in the liver, but also attenuates diabetes-induced inflammation, then contributes to ameliorate liver dysfunction. Moreover, we found that diabetes triggers the elevated RAGE in hepatocytes, and FGF1 treatment blocks it, suggesting that RAGE may be a key target during FGF1 treatment of diabetes-induced liver injury. Thus, we further confirmed the role of RAGE in FGF1 treatment of AML12 cells under high glucose condition. We found that D-ribose, a RAGE agonist, reverses the protective role of FGF1 in AML12 cells. These findings suggest that FGF1 ameliorates diabetes-induced hepatocyte apoptosis and elevated inflammation via suppressing RAGE pathway. These results suggest that RAGE may be a potential therapeutic target for the treatment of DMLD.

DL-3-n-butylphthalide ameliorates diabetes-associated cognitive decline by enhancing PI3K/Akt signaling and suppressing oxidative stress.

DL-3-n-Butylphthalide (DL-NBP), a small molecular compound extracted from the seeds of Apium graveolens Linn (Chinese celery), has been shown to exert neuroprotective effects due to its anti-inflammatory, anti-oxidative and anti-apoptotic activities. DL-NBP not only protects against ischemic cerebral injury, but also ameliorates vascular cognitive impairment in dementia patients including AD and PD. In the current study, we investigated whether and how DL-NBP exerted a neuroprotective effect against diabetes-associated cognitive decline (DACD) in db/db mice, a model of type-2 diabetes. db/db mice were orally administered DL-NBP (20, 60, 120 mg· kg-1· d-1) for 8 weeks. Then the mice were subjected to behavioral test, their brain tissue was collected for morphological and biochemical analyses. We showed that oral administration of DL-NBP significantly ameliorated the cognitive decline with improved learning and memory function in Morris water maze testing. Furthermore, DL-NBP administration attenuated diabetes-induced morphological alterations and increased neuronal survival and restored the levels of synaptic protein PSD95, synaptophysin and synapsin-1 as well as dendritic density in the hippocampus, especially at a dose of 60 mg/kg. Moreover, we revealed that DL-NBP administration suppressed oxidative stress by upregulating Nrf2/HO-1 signaling, and increased brain-derived neurotrophic factor (BDNF) expression by activating PI3K/Akt/CREB signaling in the hippocampus. These beneficial effects of DL-NBP were observed in high glucose-treated PC12 cells. Our results suggest that DL-NBP may be a potential pharmacologic agent for the treatment of DACD.

Dysregulation of Rab5-mediated endocytic pathways in Alzheimer's disease.

Increasing evidence has pointed to that dysregulation of the endo-lysosomal system is an early cellular phenotype of pathogenesis for Alzheimer's disease (AD). Rab5, a small GTPase, plays a critical role in mediating these processes. Abnormal overactivation of Rab5 has been observed in post-mortem brain samples of Alzheimer's patients as well as brain samples of mouse models of AD. Recent genome-wide association studies of AD have identified RIN3 (Ras and Rab interactor 3) as a novel risk factor for the disease. RIN3 that functions as a guanine nucleotide exchange factor for Rab5 may serve as an important activator for Rab5 in AD pathogenesis. In this review, we present recent research highlights on the possible roles of dysregulation of Rab5-mediated endocytic pathways in contributing to early pathogenesis of AD.

T-complex protein 1-ring complex enhances retrograde axonal transport by modulating tau phosphorylation.

The cytosolic chaperonin T-complex protein (TCP) 1-ring complex (TRiC) has been shown to exert neuroprotective effects on axonal transport through clearance of mutant Huntingtin (mHTT) in Huntington's disease. However, it is presently unknown if TRiC also has any effect on axonal transport in wild-type neurons. Here, we examined how TRiC impacted the retrograde axonal transport of brain-derived neurotrophic factor (BDNF). We found that expression of a single TRiC subunit significantly enhanced axonal transport of BDNF, leading to an increase in instantaneous velocity with a concomitant decrease in pauses for retrograde BDNF transport. The transport enhancing effect by TRiC was dependent on endogenous tau expression because no effect was seen in neurons from tau knockout mice. We showed that TRiC regulated the level of cyclin-dependent kinase 5 (CDK5)/p35 positively, contributing to TRiC-mediated tau phosphorylation (ptau). Expression of a single TRiC subunit increased the level of ptau while downregulation of the TRiC complex decreased ptau. We further demonstrated that TRiC-mediated increase in ptau induced detachment of tau from microtubules. Our study has thus revealed that TRiC-mediated increase in tau phosphorylation impacts retrograde axonal transport.

Exogenous fibroblast growth factor 1 ameliorates diabetes-induced cognitive decline via coordinately regulating PI3K/AKT signaling and PERK signaling.

BACKGROUND: Diabetes induces central nervous system damage, leading to cognitive decline. Fibroblast growth factor 1 (FGF1) has dual function of neuroprotection and normalizing hyperglycemia. To date, the precise mechanisms and potential treating strategies of FGF1 for diabetes-induced cognitive decline (DICD) hasn't been fully elucidated. METHODS: In this study, db/db mice were used as DICD animal model. We found that diabetes remarkably suppressed FGF1 expression in hippocampus. Thus, exogenous FGF1 had been treated for db/db mice and SH-SY5Y cells. RESULTS: FGF1 significantly ameliorates DICD with better spatial learning and memory function. Moreover, FGF1 blocked diabetes-induced morphological structure change, neuronal apoptosis and Aß1-42 deposition and synaptic dysfunction in hippocampus. But normalizing glucose may not the only contributed factor for FGF1 treating DICD with evidencing that metformin-treated db/db mice has a inferior cognitive function than that in FGF1 group. Current mechanistic study had found that diabetes inhibits cAMP-response element binding protein (CREB) activity and subsequently suppresses brain derived neurotrophic factor (BDNF) level via coordinately regulating PERK signaling and PI3K/AKT signaling in hippocampus, which were reversed by FGF1. CONCLUSION: We conclude that FGF1 exerts its neuroprotective role and normalizing hyperglycemia effect, consequently ameliorates DICD, implying FGF1 holds a great promise to develop a new treatment for DICD. Video abstract.

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception.

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFß sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF.

TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington's disease.

Corticostriatal atrophy is a cardinal manifestation of Huntington's disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagent-mediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons.

Reducing Endogenous α-Synuclein Mitigates the Degeneration of Selective Neuronal Populations in an Alzheimer's Disease Transgenic Mouse Model.

UNLABELLED: Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid ß (Aß) and microtubule associate protein tau, leading to the selective degeneration of neurons in the neocortex, limbic system, and nucleus basalis, among others. Recent studies have shown that α-synuclein (α-syn) also accumulates in the brains of patients with AD and interacts with Aß and tau, forming toxic hetero-oligomers. Although the involvement of α-syn has been investigated extensively in Lewy body disease, less is known about the role of this synaptic protein in AD. Here, we found that reducing endogenous α-syn in an APP transgenic mouse model of AD prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. Together, these results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of Aß. SIGNIFICANCE STATEMENT: Reducing endogenous α-synuclein (α-syn) in an APP transgenic mouse model of Alzheimer's disease (AD) prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. These results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of amyloid ß.

Charcot Marie Tooth 2B Peripheral Sensory Neuropathy: How Rab7 Mutations Impact NGF Signaling?

Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B) is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s) enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF) in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A "gain of toxicity" model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s) causes a "loss of function", resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons) of CMT2B are needed to precisely define the disease mechanisms.

Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling.

Retrograde trophic signaling of nerve growth factor (NGF) supports neuronal survival and differentiation. Dysregulated trophic signaling could lead to various neurological disorders. Charcot-Marie-Tooth type 2B (CMT2B) is one of the most common inherited peripheral neuropathies characterized by severe terminal axonal loss. Genetic analysis of human CMT2B patients has revealed four missense point mutations in Rab7, a small GTPase that regulates late endosomal/lysosomal pathways, but the exact pathological mechanism remains poorly understood. Here, we show that these Rab7 mutants dysregulated axonal transport and diminished the retrograde signaling of NGF and its TrkA receptor. We found that all CMT2B Rab7 mutants were transported significantly faster than Rab7(wt) in the anterograde direction, accompanied with an increased percentile of anterograde Rab7-vesicles within axons of rat E15.5 dorsal root ganglion (DRG) neurons. In PC12M cells, the CMT2B Rab7 mutants drastically reduced the level of surface TrkA and NGF binding, presumably by premature degradation of TrkA. On the other hand, siRNA knock-down of endogenous Rab7 led to the appearance of large TrkA puncta in enlarged Rab5-early endosomes within the cytoplasm, suggesting delayed TrkA degradation. We also show that CMT2B Rab7 mutants markedly impaired NGF-induced Erk1/2 activation and differentiation in PC12M cells. Further analysis revealed that CMT2B Rab7 mutants caused axonal degeneration in rat E15.5 DRG neurons. We propose that Rab7 mutants induce premature degradation of retrograde NGF-TrkA trophic signaling, which may potentially contribute to the CMT2B disease.

Impacts of H2O2, SARM1 inhibition, and high NAm concentrations on Huntington's disease laser-induced degeneration.

Axonal degeneration is a key component of neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease, and amyotrophic lateral sclerosis. Nicotinamide, an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. However, studies on nicotinamide (NAm) supplementation in humans indicate that NAm has no protective effect. Sterile alpha and toll/interleukin receptor motif-containing protein 1 (SARM1) regulates several cell responses to axonal damage and has been implicated in promoting neuronal degeneration. SARM1 inhibition seems to result in protection from neuronal degeneration while hydrogen peroxide has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type and HD dorsal root ganglion cells treated with NAm, hydrogen peroxide (H2O2), and SARM1 inhibitor DSRM-3716 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured.

Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing.

sorLA is a sorting receptor for amyloid precursor protein (APP) genetically linked to Alzheimer's disease (AD). Retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, has been implicated in APP transport because retromer deficiency leads to aberrant APP sorting and processing and levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit of the retromer complex. Accordingly, we characterized the interaction between the retromer complex and sorLA and determined the role of retromer on sorLA-dependent sorting and processing of APP. Mutations in the VPS26 binding site resulted in receptor redistribution to the endosomal network, similar to the situation seen in cells with VPS26 knockdown. The sorLA mutant retained APP-binding activity but, as opposed to the wild-type receptor, misdirected APP into a distinct non-Golgi compartment, resulting in increased amyloid processing. In conclusion, our data provide a molecular link between reduced retromer expression and increased amyloidogenesis as seen in patients with sporadic AD.

Age-induced nitrative stress decreases retrograde transport of proNGF via TrkA and increases proNGF retrograde transport and neurodegeneration via p75NTR.

Introduction: Axonal transport of pro nerve growth factor (proNGF) is impaired in aged basal forebrain cholinergic neurons (BFCNs), which is associated with their degeneration. ProNGF is neurotrophic in the presence of its receptor tropomyosin-related kinase A (TrkA) but induces apoptosis via the pan-neurotrophin receptor (p75NTR) when TrkA is absent. It is well established that TrkA is lost while p75NTR is maintained in aged BFCNs, but whether aging differentially affects transport of proNGF via each receptor is unknown. Nitrative stress increases during aging, but whether age-induced nitrative stress differentially affects proNGF transport via TrkA versus p75NTR has not yet been studied. Answering these questions is essential for developing an accurate understanding of the mechanisms contributing to age-induced loss of proNGF transport and BFCN degeneration. Methods: In this study, fluorescence microscopy was used to analyze axonal transport of quantum dot labeled proNGF in rat BFCNs in vitro. Receptor specific effects were studied with proNGF mutants that selectively bind to either TrkA (proNGF-KKE) or p75NTR (proNGF-Δ9-13). Signaling factor activity was quantified via immunostaining. Results: Young BFCNs transported proNGF-KKE but not proNGF-Δ9-13, and proNGF transport was not different in p75NTR knockout BFCNs compared to wildtype BFCNs. These results indicate that young BFCNs transport proNGF via TrkA. In vitro aging increased transport of proNGF-Δ9-13 but decreased transport of proNGF-KKE. Treatment with the nitric oxide synthase inhibitor L-NAME reduced retrograde transport of proNGF-Δ9-13 in aged BFCNs while increasing retrograde transport of proNGF-KKE but did not affect TrkA or p75NTR levels. ProNGF-Δ9-13 induced greater pro-apoptotic signaling and neurodegeneration and less pro-survival signaling relative to proNGF-KKE. Discussion: Together, these results indicate that age-induced nitrative stress decreases proNGF transport via TrkA while increasing proNGF transport via p75NTR. These transport deficits are associated with decreased survival signaling, increased apoptotic signaling, and neurodegeneration. Our findings elucidate the receptor specificity of age-and nitrative stress-induced proNGF transport deficits. These results may help to rescue the neurotrophic signaling of proNGF in aging to reduce age-induced loss of BFCN function and cognitive decline.

Hippocampal Reduction of α-Synuclein via RNA Interference Improves Neuropathology in Alzheimer's Disease Mice.

BACKGROUND: Alzheimer's disease (AD) cases are often characterized by the pathological accumulation of α-synuclein (α-syn) in addition to amyloid-ß (Aß) and tau hallmarks. The role of α-syn has been extensively studied in synucleinopathy disorders, but less so in AD. Recent studies have shown that α-syn may also play a role in AD and its downregulation may be protective against the toxic effects of Aß accumulation. OBJECTIVE: We hypothesized that selectively knocking down α-syn via RNA interference improves the neuropathological and biochemical findings in AD mice. METHODS: Here we used amyloid precursor protein transgenic (APP-Tg) mice to model AD and explore pathologic and behavioral phenotypes with knockdown of α-syn using RNA interference. We selectively reduced α-syn levels by stereotaxic bilateral injection of either LV-shRNA α-syn or LV-shRNA-luc (control) into the hippocampus of AD mice. RESULTS: We found that downregulation of α-syn results in significant reduction in the number of Aß plaques. In addition, mice treated with LV-shRNA α-syn had amelioration of abnormal microglial activation (Iba1) and astrocytosis (GFAP) phenotypes in AD mice. CONCLUSION: Our data suggests a novel link between Aß and α-syn pathology as well as a new therapeutic angle for targeting AD.

Lipid Nanoparticle Delivery of Chemically Modified NGFR100W mRNA Alleviates Peripheral Neuropathy.

Messenger RNA (mRNA) carries genetic instructions to the cell machinery for the transient production of antigens or therapeutic proteins and shows enormous potential in vaccine development, cancer immunotherapy, protein replacement therapy, and genome engineering. Here, the synthesis of chemically modified nerve growth factor mutant (NGFR100W ) mRNA through in vitro transcription is described. After the replacement of the original signal peptide sequence with the Ig Kappa leader sequence, codon-optimized NGFR100W mRNA yielded high secretion of mature NGFR100W , which promotes axon growth in PC12 cells. Using lipid nanoparticle (LNP)-delivery of N1-methylpseudouridine-modified mRNA in mice, NGFR100W -mRNA-LNPs result in the successful expression of NGFR100W protein, which significantly reduces nociceptive activity compared to that of NGFWT . This indicates that NGFR100W derived from exogenous mRNA elicited "painless" neuroprotective activity. Additionally, the therapeutic value of NGFR100W mRNA is established in a paclitaxel-induced peripheral neuropathy model by demonstrating the rapid recovery of intraepidermal nerve fibers. The results show that in vitro-transcribed mRNA has significant flexibility in sequence design and fast in vivo functional validation of target proteins. Furthermore, the results highlight the therapeutic potential of mRNA as a supplement to beneficial proteins for preventing or reversing some chronic medical conditions, such as peripheral neuropathy.