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Autophagy is a novel pathway for neurofilament protein degradation in vivo

Rao, Mala V; Darji, Sandipkumar; Stavrides, Philip H; Goulbourne, Chris N; Kumar, Asok; Yang, Dun-Sheng; Yoo, Lang; Peddy, James; Lee, Ju-Hyun; Yuan, Aidong; Nixon, Ralph A
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.
PMID: 36131358
ISSN: 1554-8635
CID: 5335462

Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques

Lee, Ju-Hyun; Yang, Dun-Sheng; Goulbourne, Chris N; Im, Eunju; Stavrides, Philip; Pensalfini, Anna; Chan, Han; Bouchet-Marquis, Cedric; Bleiwas, Cynthia; Berg, Martin J; Huo, Chunfeng; Peddy, James; Pawlik, Monika; Levy, Efrat; Rao, Mala; Staufenbiel, Mathias; Nixon, Ralph A
Autophagy is markedly impaired in Alzheimer's disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aβ/APP-βCTF selectively within enlarged de-acidified autolysosomes. In more compromised yet still intact neurons, profuse Aβ-positive autophagic vacuoles (AVs) pack into large membrane blebs forming flower-like perikaryal rosettes. This unique pattern, termed PANTHOS (poisonous anthos (flower)), is also present in AD brains. Additional AVs coalesce into peri-nuclear networks of membrane tubules where fibrillar β-amyloid accumulates intraluminally. Lysosomal membrane permeabilization, cathepsin release and lysosomal cell death ensue, accompanied by microglial invasion. Quantitative analyses confirm that individual neurons exhibiting PANTHOS are the principal source of senile plaques in amyloid precursor protein AD models.
PMID: 35654956
ISSN: 1546-1726
CID: 5236172

Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo

Lee, Ju-Hyun; Rao, Mala V; Yang, Dun-Sheng; Stavrides, Philip; Im, Eunju; Pensalfini, Anna; Huo, Chunfeng; Sarkar, Pallabi; Yoshimori, Tamotsu; Nixon, Ralph A
Autophagy-lysosome pathway (ALP) disruption is considered pathogenic in multiple neurodegenerative diseases; however, current methods are inadequate to investigate macroautophagy/autophagy flux in brain in vivo and its therapeutic modulation. Here, we describe a novel autophagy reporter mouse (TRGL6) stably expressing a dual-fluorescence-tagged LC3 (tfLC3, mRFP-eGFP-LC3) by transgenesis selectively in neurons. The tfLC3 probe distributes widely in the central nervous system, including spinal cord. Expression levels were similar to endogenous LC3 and induced no detectable ALP changes. This ratiometric reporter registers differential pH-dependent changes in color as autophagosomes form, fuse with lysosomes, acidify, and degrade substrates within autolysosomes. We confirmed predicted changes in neuronal autophagy flux following specific experimental ALP perturbations. Furthermore, using a third fluorescence label in TRGL6 brains to identify lysosomes by immunocytochemistry, we validated a novel procedure to detect defective autolysosomal acidification in vivo. Thus, TRGL6 mice represent a unique tool to investigate in vivo ALP dynamics in specific neuron populations in relation to neurological diseases, aging, and disease modifying agents. Abbreviations: ACTB: actin, beta; AD: Alzheimer disease; AL: autolysosomes; ALP: autophagy-lysosome pathway; AP: autophagosome; APP: amyloid beta (Abeta) precursor protein; ATG5: autophagy related 5; ATG7: autophagy related 7; AV: autophagic vacuoles; CNS: central nervous system; CTSD: cathepsin D; CQ: chloroquine; DMEM: Dulbecco's modified Eagle's medium; GFP: green fluorescent protein; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2/GATE16: gamma-aminobutyric acid (GABA) receptor-associated protein-like 2; ICC: immunocytochemistry; ICV: intra-cerebroventricular; LAMP2: lysosomal-associated membrane protein 2; Leup: leupeptin; LY: lysosomes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RFP: red fluorescent protein; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; tfLC3: mRFP-eGFP-LC3; TRGL6: Thy1 mRFP eGFP LC3-line 6; PCR: polymerase chain reaction; PD: Parkinson disease.
PMID: 30269645
ISSN: 1554-8635
CID: 3631072

Neurofilament light interaction with GluN1 modulates neurotransmission and schizophrenia-associated behaviors

Yuan, Aidong; Sershen, Henry; Basavarajappa, Balapal S; Smiley, John F; Hashim, Audrey; Bleiwas, Cynthia; Berg, Martin; Guifoyle, David N; Subbanna, Shivakumar; Darji, Sandipkumar; Kumar, Asok; Rao, Mala V; Wilson, Donald A; Julien, Jean-Pierre; Javitt, Daniel C; Nixon, Ralph A
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.
PMID: 30143609
ISSN: 2158-3188
CID: 3246612

Differential induction of antibody responses to V1V2 and V3 of HIV envelope GP120 by immune complex vaccines [Meeting Abstract]

Hioe, C; Kumar, R; Upadhyay, C; Jan, Peer M; Fox, A; Itri, V; Peachman, K; Rao, M; Liu, L; Lo, N; Tuen, M; Jiang, X; Kong, X -P; Zolla-Pazner, S
Background: To be effective, prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope (Env). Although HIV Env is renowned for its variability, it also contains regions that are conserved, albeit poorly immunogenic. To direct the Ab response toward the more conserved Env sites, we utilized immune complex vaccines made of the Env protein gp120 or gp140 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously demonstrated the ability of gp120/mAb immune complexes to enhance the elicitation of V3 Abs; however, Ab response to other regions, especially the V1V2 domain recently identified as an important target for protective Abs against HIV, has not been studied; neither have immune complex vaccines with non-B-subtype Env.
Method(s): This study compared immunogenicity of subtypes B (JRFL), C (CN54), and CRF-01.AE (A244) Env in complex with selected gp120-specific mAbs in mice.
Result(s): Immunization with the complexes elicited comparable serum IgG titers against Env, but a marked skewing toward V1V2 or V3 was evident and dependent on the Env strain and the specificity of the mAb used to form the complexes. Compared with gp120JRFL, immunization with gp120JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited greater IgG titers against V3 from subtypes A, B, and C. Epitope mapping revealed a shift toward a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp140CN54/mAb and gp120A244/mAb complexes. Notably, gp120A244/ mAb complexes induced higher levels of V1V2 Abs, while stimulating weak or strain-specific V3 Abs. Along with altered immunogenicity, allosteric and antigenic changes were detected on these complexes, indicating that mAb interaction induces alterations on the Env surface that modify its immunogenic property.
Conclusion(s): Immune complex vaccines may be useful to shape Ab responses toward Env sites of interest
ISSN: 1931-8405
CID: 3528212

Neurofilaments and Neurofilament Proteins in Health and Disease

Yuan, Aidong; Rao, Mala V; Veeranna; Nixon, Ralph A
SUMMARYNeurofilaments (NFs) are unique among tissue-specific classes of intermediate filaments (IFs) in being heteropolymers composed of four subunits (NF-L [neurofilament light]; NF-M [neurofilament middle]; NF-H [neurofilament heavy]; and alpha-internexin or peripherin), each having different domain structures and functions. Here, we review how NFs provide structural support for the highly asymmetric geometries of neurons and, especially, for the marked radial expansion of myelinated axons crucial for effective nerve conduction velocity. NFs in axons extensively cross-bridge and interconnect with other non-IF components of the cytoskeleton, including microtubules, actin filaments, and other fibrous cytoskeletal elements, to establish a regionally specialized network that undergoes exceptionally slow local turnover and serves as a docking platform to organize other organelles and proteins. We also discuss how a small pool of oligomeric and short filamentous precursors in the slow phase of axonal transport maintains this network. A complex pattern of phosphorylation and dephosphorylation events on each subunit modulates filament assembly, turnover, and organization within the axonal cytoskeleton. Multiple factors, and especially turnover rate, determine the size of the network, which can vary substantially along the axon. NF gene mutations cause several neuroaxonal disorders characterized by disrupted subunit assembly and NF aggregation. Additional NF alterations are associated with varied neuropsychiatric disorders. New evidence that subunits of NFs exist within postsynaptic terminal boutons and influence neurotransmission suggests how NF proteins might contribute to normal synaptic function and neuropsychiatric disease states.
PMID: 28373358
ISSN: 1943-0264
CID: 2519392

Calpastatin inhibits motor neuron death and increases survival of hSOD1 mice

Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain overactivation in hSOD1G93A transgenic (Tg) mice in vivo by neuron specific overexpression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that overexpression of CAST in hSOD1G93A mice, which lowered calpain activation to levels comparable to WT mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1G93A /CAST mice compared to littermate hSOD1G93A mice is delayed, which accounts for their longer time of survival. We also find that neuronal overexpression of CAST in hSOD1G93A transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model
PMID: 26756888
ISSN: 1471-4159
CID: 1912592

Correction to: Impact of nonoptimal intakes of saturated, polyunsaturated, and trans fat on global burdens of coronary heart disease. [J Am Heart Assoc. (2016) 5, e002891.] Doi:10.1161/JAHA.115.002891

Wang, Q; Afshin, A; Yakoob, M Y; Singh, G M; Rehm, C D; Khatibzadeh, S; Micha, R; Shi, P; Mozaffarian, D; Ezzati, M; Fahimi, S; Wirojratana, P; Powles, J; Elmadfa, I; Rao, M; Alpert, W; Lim, S S; Engell, R E; Andrews, K G; Abbott, P A; Abdollahi, M; Abeya, Gilardon E O; Ahsan, H; Al, Nsour M A A; Al-Hooti, S N; Arambepola, C; Fernando, D N; Barennes, H; Barquera, S; Baylin, A; Becker, W; Bjerregaard, P; Bourne, L T; Capanzana, M V; Castetbon, K; Chang, H -Y; Chen, Y; Cowan, M J; Riley, L M; De, Henauw S; Ding, E L; Duante, C A; Duran, P; Barbieri, H E; Farzadfar, F; Hadziomeragic, A F; Fisberg, R M; Forsyth, S; Garriguet, D; Gaspoz, J -M; Gauci, D; Calleja, N; Ginnela, B N V; Guessous, I; Gulliford, M C; Hadden, W; Haerpfer, C; Hoffman, D J; Houshiar-Rad, A; Huybrechts, I; Hwalla, N C; Ibrahim, H M; Inoue, M; Jackson, M D; Johansson, L; Keinan-Boker, L; Kim, C -I; Koksal, E; Lee, H -J; Li, Y; Lipoeto, N I; Ma, G; Mangialavori, G L; Matsumura, Y; McGarvey, S T; Fen, C M; Monge-Rojas, R A; Musaiger, A O; Nagalla, B; Naska, A; Ocke, M C; Oltarzewski, M; Szponar, L; Orfanos, P; Ovaskainen, M -L; Tapanainen, H; Pan, W -H; Panagiotakos, D B; Pekcan, G A; Petrova, S; Piaseu, N; Pitsavos, C; Posada, L G; Sanchez-Romero, L M; Selamat, R B T; Sharma, S; Sibai, A M; Sichieri, R; Simmala, C; Steingrimsdottir, L; Swan, G; Sygnowska, E H; Templeton, R; Thanopoulou, A; Thorgeirsdottir, H; Thorsdottir, I; Trichopoulou, A; Tsugane, S; Turrini, A; Vaask, S; van, Oosterhout C; Veerman, J L; Verena, N; Waskiewicz, A; Zaghloul, S; Zajkas, G
In the article by Wang et al, "Impact of Nonoptimal Intakes of Saturated, Polyunsaturated, and Trans Fat on Global Burdens of Coronary Heart Disease," which published online January 20, 2016, and appeared in the January 2016 issue of the journal (J Am Heart Assoc. 2016;5:e002891 doi:10.1161/ JAHA.115.002891), the full list of the Global Burden of Diseases Nutrition and Chronic Diseases Expert Group (NutriCoDE) group were erroneously listed as authors in the HTML version of the article. The publisher regrets the error. The online version of the article has been updated and is available at e002891.
ISSN: 2047-9980
CID: 4548832

Neurofilament subunits are integral components of synapses and modulate neurotransmission and behavior in vivo

Yuan, A; Sershen, H; Veeranna; Basavarajappa, B S; Kumar, A; Hashim, A; Berg, M; Lee, J-H; Sato, Y; Rao, M V; Mohan, P S; Dyakin, V; Julien, J-P; Lee, V M-Y; Nixon, R A
Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (alpha-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.
PMID: 25869803
ISSN: 1476-5578
CID: 1684462

Functions of neurofilaments in synapses

Yuan, A; Sershen, H; Veeranna; Basavarajappa, B S; Kumar, A; Hashim, A; Berg, M; Lee, J-H; Sato, Y; Rao, M V; Mohan, P S; Dyakin, V; Julien, J-P; Lee, V M-Y; Nixon, R A
PMID: 26201270
ISSN: 1476-5578
CID: 1683992