Panhelainen, A. | bioRxiv Science

bioRxiv2019

Gene Knock Up via 3’UTR editing to study gene function in vivo

Currently available genetic tools do not allow researchers to upregulate ( Knock Up) the levels of a given protein while retaining its cell-type-specific regulation. As a result, we have limited ability to develop overexpression-related disease models, to study the contribution of single genes in diseases caused by copy number variations and to identify disease pathways for drug targets. Here we develop two approaches for endogenous gene upregulation: conditional Knock Up (cKU) utilizing the Cre/lox system, and CRISPR-Cas9 mediated gene Knock Up (KU) in wild-type mouse embryos and human cells. Using glial cell line derived neurotrophic factor (GDNF) as a proof of concept, we show that both approaches resulted in upregulation of endogenous GDNF levels without disturbing Gdnfs expression pattern. Furthermore, CNS-specific GDNF cKU resulted in dopaminergic abnormalities and schizophrenia-like phenotypes. Our results suggest that gene Knock Up can reveal unknown gene functions and provide novel entry points for studying neurological disease.

neuroscience↗

bioRxiv2019

GDNF/RET signaling pathway activation eliminates Lewy Body pathology in midbrain dopamine neurons

Neurodegenerative diseases are associated with proteostasis disturbances and accumulation of fibrillar proteins into insoluble aggregates. Progressive age-related degeneration of dopamine neurons is a primary cause of motor dysfunctions in Parkinsons disease (PD) and substantial evidence supports critical involvement of -synuclein (-syn) in the etiology of PD. -syn is a cytosolic protein present in high concentrations in pre-synaptic neuronal terminals and a primary constituent of intracellular protein aggregates known as Lewy Neurites or Lewy Bodies. Progression of Lewy pathology is a characteristic feature in the PD brains caused by the prion-like self-templating properties of misfolded -syn. Modelling Lewy pathology progression with application of exogenously prepared -syn preformed fibrils, we discovered that glial cell line-derived neurotrophic factor (GDNF) prevented formation of -syn aggregates in dopamine neurons in culture and in vivo after viral vector expression of GDNF. These effects were abolished by CRISPR/Cas9-mediated deletion of receptor tyrosine kinase Ret, the major GDNF signaling pathway. Similar to GDNF, expression of mutated constitutively active RET (RET_MEN2B) was able to protect dopamine neurons. GDNF protection against -syn pathology progression was abolished by Src and attenuated by Akt pathway inhibitors. For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded -syn propagation in dopamine neurons, uncovered underlying receptor and intracellular signaling pathways. These results for the first time demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=145 HEIGHT=200 SRC=\"FIGDIR/small/752899v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (57K):\norg.highwire.dtl.DTLVardef@1f5109dorg.highwire.dtl.DTLVardef@15e4a2org.highwire.dtl.DTLVardef@15571c8org.highwire.dtl.DTLVardef@a10f7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗