Biology subjects

Rao, N. R.

Publications and source records attributed to Rao, N. R..

4 recordsLinked to original sources

bioRxiv2024

The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity

The degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in the DNAJC7 gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X in DNAJC7 exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.

neuroscience↗

bioRxiv2024

Levetiracetam prevents Aβ42 production through SV2a-dependent modulation of App processing in Alzheimer's disease models

In Alzheimers disease (AD), amyloid-beta (A{beta}) peptides are produced by proteolytic cleavage of the amyloid precursor protein (APP), which can occur during synaptic vesicle (SV) cycling at presynapses. Precisely how amyloidogenic APP processing may impair presynaptic proteostasis and how to therapeutically target this process remains poorly understood. Using App knock-in mouse models of early A{beta} pathology, we found proteins with hampered degradation accumulate at presynaptic sites. At this mild pathological stage, amyloidogenic processing leads to accumulation of A{beta}42 inside SVs. To explore if targeting SVs modulates A{beta} accumulation, we investigated levetiracetam (Lev), a SV-binding small molecule drug that has shown promise in mitigating AD-related pathologies despite its mechanism of action being unclear. We discovered Lev reduces A{beta}42 levels by decreasing amyloidogenic processing of APP in a SV2a-dependent manner. Lev corrects SV protein levels and cycling, which results in increased surface localization of APP, where it favors processing via the non-amyloidogenic pathway. Using metabolic stable isotopes and mass spectrometry we confirmed that Lev prevents the production of A{beta}42 in vivo. In transgenic mice with aggressive pathology, electrophysiological and immunofluorescent microscopy analyses revealed that Lev treatment reduces SV cycling and minimizes synapse loss. Finally, we found that human Down syndrome brains with early A{beta} pathology, have elevated levels of presynaptic proteins, confirming a comparable presynaptic deficit in human brains. Taken together, we report a mechanism that highlights the therapeutic potential of Lev to modify the early stages of AD and represent a promising strategy to prevent A{beta}42 pathology before irreversible damage occurs. One Sentence SummaryWe discovered that the SV-binding drug levetiracetam prevents A{beta}42 production by modulating SV cycling which alters APP localization and thus proteolytic processing, highlighting its therapeutic potential for AD.

neuroscience↗

bioRxiv2023

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity

BackgroundThe accumulation of amyloid beta (A{beta}) peptides in fibrils is prerequisite for Alzheimers disease (AD). Our understanding of the proteins that promote A{beta} fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. MethodsTo investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of A{beta} immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic A{beta}42 flies, and A{beta}42 seeded cultured neurons. ResultsAmyloid fibrils are primarily composed by A{beta}42 and unexpectedly harbor A{beta}38 but generally lack A{beta}40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 17 new amyloid-associated proteins. Notably, we confirmed 126 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic A{beta}42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Eight RNAi lines suppressed and 11 enhanced A{beta}42 toxicity. ConclusionsThese discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

neuroscience↗

bioRxiv2021

Levetiracetam treatment normalizes levels of the presynaptic endocytosis machinery and restores non-amyloidogenic APP processing in App knock-in mice.

Increasing evidence indicates that toxic amyloid-beta (A{beta}) peptides, produced by sequential proteolytic cleavage of the Amyloid Precursor Protein (APP), induce neuronal circuit hyperexcitability in the early stages of Alzheimers disease (AD). As a result, treatments that modulate this early excitatory/inhibitory imbalance could act as potential AD therapies. Levetiracetam, an atypical antiepileptic drug, has garnered recent interest, despite the mechanism(s) of action remaining elusive. In this study, we set out to identify the pathways and mechanisms primarily affected by levetiracetam in diseased brains of amyloid pathology. Using the App knock-in mouse models and multiplexed TMT-quantitative mass spectrometry-based proteomic analysis to determine how levetiracetam affects the proteome, our findings demonstrate that levetiracetam treatment selectively normalizes levels of presynaptic endocytosis proteins and is capable of lowering A{beta}42 levels by altering APP processing. These novel findings demonstrate a mechanism of action for how levetiracetam lowers A{beta}42 production.

neuroscience↗