Name: Lentiviral-Induced Striatal Pathology as a Preclinical Model of Polyglutamine Spinocerebellar Ataxias (Video) | JoVE Journal
Uploaded: 2026-03-13T14:30:00.000+00:00
Duration: 9 min 12 s
Description: Discover lentiviral-based striatal modeling for polyglutamine spinocerebellar ataxias using stereotaxic injection in C57BL6J mice. This method enables region-specific expression of expanded ataxin-2 to study disease mechanisms and evaluate therapeutic interventions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Cl&#233;vio N&#243;brega&#39;s laboratory is funded by the Cure CSB project, by the Viljem Julijan Association for Children with Rare Diseases (Slovenia), and by the Algarve Biomedical Center Research Institute (ABC-Ri). Some of the authors of this paper have a PhD fellowship funded by Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT), with the following references: Tiago Moreira-Gomes (2022.11008.BD), In&#234;s T Afonso (2022.10161.BD), and Rafael G Costa (2022. 11973.BD).

<ol><li>Afonso-Reis, R., Afonso, I. T., Nóbrega, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+Status+of+Gene+Therapy+Research+in+Polyglutamine+Spinocerebellar+Ataxias%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Current Status of Gene Therapy Research in Polyglutamine Spinocerebellar Ataxias.</a> Int J Mol Sci. 22 (8), 4249(2021).</li>
<li>Durr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autosomal+dominant+cerebellar+ataxias%3A+polyglutamine+expansions+and+beyond%5BTitle%5D)+AND+%22Lancet+Neurol%22%5BJournal%5D)">Autosomal dominant cerebellar ataxias: polyglutamine expansions and beyond.</a> Lancet Neurol. 9 (9), 885-894 (2010).</li>
<li>McIntosh, C. S., Li, D., Wilton, S. D., Aung-Htut, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyglutamine+Ataxias%3A+Our+Current+Molecular+Understanding+and+What+the+Future+Holds+for+Antisense+Therapies%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Polyglutamine Ataxias: Our Current Molecular Understanding and What the Future Holds for Antisense Therapies.</a> Biomedicines. 9 (11), 1499(2021).</li>
<li>Henriques, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viral-based+animal+models+in+polyglutamine+disorders%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Viral-based animal models in polyglutamine disorders.</a> Brain. 147 (4), 1166-1189 (2024).</li>
<li>Doyle, A., McGarry, M. P., Lee, N. A., Lee, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Construction+of+Transgenic+and+Gene+Knockout%2FKnockin+Mouse+Models+of+Human+Disease%5BTitle%5D)+AND+%22Transg+Res%22%5BJournal%5D)">The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease.</a> Transg Res. 21 (2), 327-349 (2012).</li>
<li>Tello, J. A., Williams, H. E., Eppler, R. M., Steinhilb, M. L., Khanna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+Models+of+Neurodegenerative+Disease%3A+Recent+Advances+in+Fly+Highlight+Innovative+Approaches+to+Drug+Discovery%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">Animal Models of Neurodegenerative Disease: Recent Advances in Fly Highlight Innovative Approaches to Drug Discovery.</a> Front Mol Neurosci. 15, 883358(2022).</li>
<li>Moreira-Gomes, T., Nóbrega, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+gene+therapy+with+lentiviral+vectors+for+spinocerebellar+ataxias%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Development of a gene therapy with lentiviral vectors for spinocerebellar ataxias.</a> Methods Mol Biol. 2974, 215-225 (2026).</li>
<li>Parr-Brownlie, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+vectors+as+tools+to+understand+central+nervous+system+biology+in+mammalian+model+organisms%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms.</a> Front Mol Neurosci. 8, 14(2015).</li>
<li>Zwi-Dantsis, L., Mohamed, S., Massaro, G., Moeendarbary, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adeno-Associated+Virus+Vectors%3A+Principles%2C+Practices%2C+and+Prospects+in+Gene+Therapy%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Adeno-Associated Virus Vectors: Principles, Practices, and Prospects in Gene Therapy.</a> Viruses. 17 (2), 239(2025).</li>
<li>Moreira-Gomes, T., Nóbrega, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+the+disruption+of+RNA+metabolism+to+the+targeting+of+RNA-binding+proteins%3A+The+case+of+polyglutamine+spinocerebellar+ataxias%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">From the disruption of RNA metabolism to the targeting of RNA-binding proteins: The case of polyglutamine spinocerebellar ataxias.</a> J Neurochem. 168 (8), 1442-1459 (2024).</li>
<li>Matsuzaki, Y., Oue, M., Hirai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+a+neurodegenerative+disease+mouse+model+using+lentiviral+vectors+carrying+an+enhanced+synapsin+I+promoter%5BTitle%5D)+AND+%22J+Neurosci+Meth%22%5BJournal%5D)">Generation of a neurodegenerative disease mouse model using lentiviral vectors carrying an enhanced synapsin I promoter.</a> J Neurosci Meth. 223, 133-143 (2014).</li>
<li>Marcelo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autophagy+in+Spinocerebellar+ataxia+type+2%2C+a+dysregulated+pathway%2C+and+a+target+for+therapy%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Autophagy in Spinocerebellar ataxia type 2, a dysregulated pathway, and a target for therapy.</a> Cell Death Dis. 12 (12), 1117(2021).</li>
<li>Alves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Striatal+and+nigral+pathology+in+a+lentiviral+rat+model+of+Machado-Joseph+disease%5BTitle%5D)+AND+%22Human+Mol+Genet%22%5BJournal%5D)">Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease.</a> Human Mol Genet. 17 (14), 2071-2083 (2008).</li>
<li>Alves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+vector-mediated+overexpression+of+mutant+ataxin-7+recapitulates+SCA7+pathology+and+promotes+accumulation+of+the+FUS%2FTLS+and+MBNL1+RNA-binding+proteins%5BTitle%5D)+AND+%22Mol+Neurodegenerat%22%5BJournal%5D)">Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins.</a> Mol Neurodegenerat. 11 (1), 58(2016).</li>
<li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+lentiviral+vector+titration+methods%5BTitle%5D)+AND+%22BMC+Biotechnol%22%5BJournal%5D)">Comparison of lentiviral vector titration methods.</a> BMC Biotechnol. 6 (1), 34(2006).</li>
<li>Arrasate, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+Characterization+of+a+Stable+Producer+Cell+Line+Generation+Platform+for+the+Manufacturing+of+Clinical-Grade+Lentiviral+Vectors%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Establishment and Characterization of a Stable Producer Cell Line Generation Platform for the Manufacturing of Clinical-Grade Lentiviral Vectors.</a> Biomedicines. 12 (10), 2265(2024).</li>
<li>Nóbrega, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+Mutant+Ataxin-3+in+Mouse+Cerebellum+Induces+Ataxia+and+Cerebellar+Neuropathology%5BTitle%5D)+AND+%22Cerebellum%22%5BJournal%5D)">Overexpression of Mutant Ataxin-3 in Mouse Cerebellum Induces Ataxia and Cerebellar Neuropathology.</a> Cerebellum. 12 (4), 441-455 (2013).</li>
<li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+Mouse+Brain+in+Stereotaxic+Coordinates%22">The Mouse Brain in Stereotaxic Coordinates</a>. , Compact Second Edition, Gulf Professional Publishing. (2004).</li>
<li>Bateup, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+subclasses+of+medium+spiny+neurons+differentially+regulate+striatal+motor+behaviors%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors.</a> Proc Natl Acad Sci U S A. 107 (33), 14845-14850 (2010).</li>
<li>Nóbrega, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+hallmarks+of+neurodegeneration+in+polyglutamine+spinocerebellar+ataxias%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Molecular hallmarks of neurodegeneration in polyglutamine spinocerebellar ataxias.</a> Cell Death Dis. 16 (1), 826(2025).</li>
<li>Murakami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV-based+gene+therapy+ameliorated+CNS-specific+GPI+defect+in+mouse+models%5BTitle%5D)+AND+%22Mol+Ther+Meth+Clin+Dev%22%5BJournal%5D)">AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models.</a> Mol Ther Meth Clin Dev. 32 (1), 101176(2024).</li>
<li>Wada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV-based+gene+therapy+ameliorates+neurological+deficits+in+a+mouse+model+of+childhood-onset+neurodegeneration+with+cerebellar+atrophy%5BTitle%5D)+AND+%22Mol+Ther+J+Am+Soc+Gene+Ther%22%5BJournal%5D)">AAV-based gene therapy ameliorates neurological deficits in a mouse model of childhood-onset neurodegeneration with cerebellar atrophy.</a> Mol Ther J Am Soc Gene Ther. 33 (11), 5829-5839 (2025).</li>
<li>Konno, A., Shinohara, Y., Hirai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+Spinocerebellar+Ataxia+Type+3+Model+Mice+by+Intravenous+Injection+of+AAV-PHP.B+Vectors%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Production of Spinocerebellar Ataxia Type 3 Model Mice by Intravenous Injection of AAV-PHP.B Vectors.</a> Int J Mol Sci. 25 (13), 7205(2024).</li>
<li>Jansen-West, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+PolyQ-ATXN3+Levels+Associate+With+Cerebellar+Degeneration+and+Behavioral+Abnormalities+in+a+New+AAV-Based+SCA3+Mouse+Model%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Plasma PolyQ-ATXN3 Levels Associate With Cerebellar Degeneration and Behavioral Abnormalities in a New AAV-Based SCA3 Mouse Model.</a> Front Cell Dev Biol. 10, 863089(2022).</li>
<li>Nóbrega, C., Mendonça, L., Matos, C. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aN%C3%B3brega%2C+C+%22A+Handbook+of+Gene+and+Cell+Therapy%22">A Handbook of Gene and Cell Therapy</a>. , Springer International Publishing. Cham. (2020).</li>
<li>Thomas, C. E., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Progress and problems with the use of viral vectors for gene therapy.</a> Nat Rev Genet. 4 (5), 346-358 (2003).</li>
<li>Schambach, A., Zychlinski, D., Ehrnstroem, B., Baum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biosafety+Features+of+Lentiviral+Vectors%5BTitle%5D)+AND+%22Human+Gene+Ther%22%5BJournal%5D)">Biosafety Features of Lentiviral Vectors.</a> Human Gene Ther. 24 (2), 132-142 (2013).</li>
<li>de Almeida, L. P., Ross, C. A., Zala, D., Aebischer, P., Déglon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral-Mediated+Delivery+of+Mutant+Huntingtin+in+the+Striatum+of+Rats+Induces+a+Selective+Neuropathology+Modulated+by+Polyglutamine+Repeat+Size%2C+Huntingtin+Expression+Levels%2C+and+Protein+Length%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Lentiviral-Mediated Delivery of Mutant Huntingtin in the Striatum of Rats Induces a Selective Neuropathology Modulated by Polyglutamine Repeat Size, Huntingtin Expression Levels, and Protein Length.</a> J Neurosci. 22 (9), 3473-3483 (2002).</li>
<li>Fisher, E. M. C., Bannerman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+neurodegeneration%3A+Know+your+question%2C+know+your+mouse%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Mouse models of neurodegeneration: Know your question, know your mouse.</a> Sci Transl Med. 11 (493), eaaq1818(2019).</li>
<li>Bii, V. M., Collins, C. P., Hocum, J. D., Trobridge, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication-incompetent+gammaretroviral+and+lentiviral+vector-based+insertional+mutagenesis+screens+identify+prostate+cancer+progression+genes%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Replication-incompetent gammaretroviral and lentiviral vector-based insertional mutagenesis screens identify prostate cancer progression genes.</a> Oncotarget. 9 (21), 15451-15463 (2018).</li>
<li>Alves-Cruzeiro, J. M. D. C., Mendonça, L., Pereira de Almeida, L., Nóbrega, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+Dysfunctions+and+Neuropathology+in+Mouse+Models+of+Spinocerebellar+Ataxia+Type+2%3A+A+Comprehensive+Review%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Motor Dysfunctions and Neuropathology in Mouse Models of Spinocerebellar Ataxia Type 2: A Comprehensive Review.</a> Front Neurosci. 10, 572(2016).</li>
</ol>

The authors declare no competing interests.

This article describes the generation of a striatal lentiviral mouse model for a polyQ SCA. It also describes&#160;the production of high-titer lentiviral vectors encoding the wild-type and expanded form of the causative protein, and the bilateral injection of the LVs into the mouse striatum by stereotaxic surgery. Additionally, it describes the brain collection procedure, the histological processing, and the image-based quantification of disease hallmarks. Together, these methods enable the creation of a lentiv...

Polyglutamine spinocerebellar ataxias (polyQ SCAs) are a group of six autosomal dominant neurodegenerative disorders caused by CAG trinucleotide repeat expansions in various causative genes, resulting in toxic polyglutamine (polyQ) tracts within encoded proteins1. Among them, the most frequent polyQ SCAs are SCA3/Machado-Joseph disease and SCA22. These disorders primarily affect neuronal cells in specific brain regions, including the cerebellum and brainstem, as well as the striatum and other areas. Consequently, they cause progressive motor incoordination, gait disturbances, severe neurodegeneration, and premature death3.
Multiple animal models have been developed to study polyQ SCAs and to test therapeutic approaches. Transgenic mice, knock-in lines, and conditional expression mouse models have been used to elucidate the molecular basis of disease progression and the cellular pathways involved4,5. However, generating a new disease animal model is time-consuming and costly, and it is often not suitable for studying brain regional involvement in disease pathogenesis6.
Lentiviral (LV)-based mouse models offer a complementary alternative to conventional models, with reduced cost and the possibility to study specific regions of the brain7,8. Lentivirus vectors allow the delivery of large transgenes (up to 8 kb), leading to a rapid induction of pathology in defined brain regions without the need for germline modification, and they can be used in animals of different ages. The direct injection of lentiviral vectors into the target brain region can achieve region-specific expression of pathogenic or therapeutic genes with reduced immunogenicity compared to other viral vectors4. Moreover, postnatal delivery avoids developmental compensation to investigate disease mechanisms under conditions that more closely resemble human onset9. Expression levels can be titrated by adjusting viral dose, producing phenotypes of controlled severity and facilitating modeling of graded disease progression. Additionally, this method allows cell-type-specific expression dependent on the chosen promoter4,7.
Several studies have shown the practicality and usefulness of LV-based mouse models for the modeling of polyQ SCAs10,11,12,13. We and others have developed LV-based models for SCA111, SCA212, SCA311,12, and SCA714, constituting platforms for rapid, region-specific expression or silencing of disease genes while reproducing key aspects of neuropathology.
In summary, the lentiviral approach described here provides a versatile and efficient tool for modeling the pathogenesis of polyQ SCAs. By overcoming some of the major drawbacks of current animal models, this method accelerates proof-of-concept studies and provides greater control over disease onset, localization, and severity - features that make it highly suitable for therapeutic screening and the study of molecular mechanisms. Here, we describe a reproducible method to generate and characterize a striatal lentiviral model of polyglutamine spinocerebellar ataxia, enabling detailed analysis of early pathogenic events and evaluation of candidate therapeutics.

<table><tbody><tr><td>0.9% NaCl</td><td>Local supplier</td><td>&amp;mdash;</td><td>Used for preparation of anesthesia and antagonist mixes</td></tr><tr><td>100 mm tissue culture dishes</td><td>SPL Life Sciences</td><td>11090</td><td></td></tr><tr><td>ABC kit (Avidin-Biotin Complex)</td><td>VECTASTAIN Elite</td><td></td><td></td></tr><tr><td>Atipamezole (5.0 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Antagonist of medetomidine</td></tr><tr><td>Benchtop centrifuge</td><td>-</td><td>-</td><td></td></tr><tr><td>Biosafety cabinet, Class II (BSL-2 compliant)</td><td>-</td><td>-</td><td></td></tr><tr><td>Biotinylated secondary antibody</td><td>-</td><td>-</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>NZYTech</td><td>MB47002</td><td></td></tr><tr><td>Brain sections (e.g., 30&amp;ndash;40 &amp;micro;m coronal sections) immunostained for DARPP-32</td><td>-</td><td>-</td><td></td></tr><tr><td>Cell Counter plugin (FIJI)</td><td>Built-in / ImageJ</td><td>&amp;mdash;</td><td>Tool for manual aggregate counting</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator (37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;, humidified)</td><td>-</td><td>-</td><td></td></tr><tr><td>Computer workstation</td><td>Local supplier</td><td>&amp;mdash;</td><td>Used for image processing and software execution</td></tr><tr><td>Conical ultracentrifuge tubes</td><td>Beckman Coulter</td><td>358126</td><td></td></tr><tr><td>Cotton swabs</td><td>Local supplier</td><td>&amp;mdash;</td><td>Used for periosteum removal</td></tr><tr><td>Cryostat with specimen disk holder</td><td>-</td><td>-</td><td></td></tr><tr><td>DAB (Diaminobenzidine)</td><td>-</td><td>-</td><td></td></tr><tr><td>Digital stereotaxic coordinate reader</td><td>Stoelting/WPI</td><td>51725</td><td>Digital display for coordinate measurement</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM, high glucose)</td><td>Gibco/Thermo Fisher</td><td>11-965-092</td><td></td></tr><tr><td>Ethanol 70, 96 and 100%</td><td>-</td><td>-</td><td></td></tr><tr><td>Ethanol, 70%</td><td>Local supplier</td><td>&amp;mdash;</td><td>For scalp disinfection</td></tr><tr><td>Fentanyl (0.05 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Anesthetic component</td></tr><tr><td>Fetal Bovine Serum (FBS), heat-inactivated</td><td>Gibco/Thermo Fisher</td><td>10500064</td><td></td></tr><tr><td>FIJI (ImageJ distribution)</td><td>NIH / ImageJ</td><td>https://fiji.sc/</td><td>Open-source software for manual quantification of aggregates</td></tr><tr><td>Fine forceps (for toe pinch)</td><td>Local supplier</td><td>&amp;mdash;</td><td>For anesthesia depth monitoring</td></tr><tr><td>Flumazenil (0.1 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Antagonist of midazolam</td></tr><tr><td>Forceps and scalpel</td><td>Local supplier</td><td>&amp;mdash;</td><td>For periosteum removal and skull exposure</td></tr><tr><td>Hamilton&amp;nbsp;700/1700 Series Microliter/Gastight Syringes</td><td>Hamilton / Fisher Sci.</td><td>7653-01/10550203</td><td>For viral solution loading</td></tr><tr><td>Hamilton&amp;nbsp;Small Hub Removable Needles (26G)</td><td>Hamilton / Fisher Sci.</td><td>7804-03/ 10698205</td><td>For viral delivery</td></tr><tr><td>Heating pad (for rodents)</td><td>Kent Scientific / SOMNI</td><td>e.g. 04-777-177</td><td>For maintaining body temperature</td></tr><tr><td>HEK 293T cells (&amp;le; passage 10, mycoplasma-free)</td><td>ATCC or equivalent</td><td>CRL-3216</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>-</td><td>-</td><td></td></tr><tr><td>IHCAggreCount software</td><td>Supplied upon demand</td><td>N/A</td><td>Proprietary in-house software for semi-automated detection and quantification of aggregates</td></tr><tr><td>Inverted microscope (for cell confluency)</td><td>-</td><td>-</td><td></td></tr><tr><td>Laboratory vortex mixer</td><td>-</td><td>-</td><td></td></tr><tr><td>Lentiviral vectors (WT and mutant)</td><td>In-house / Core facility</td><td>N/A</td><td>Viral solution for injection</td></tr><tr><td>L-glutamine (200 mM)</td><td>Gibco/Thermo Fisher</td><td>25030081</td><td></td></tr><tr><td>Light microscope with camera (brightfield)</td><td>-</td><td>-</td><td></td></tr><tr><td>Medetomidine (1.0 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Anesthetic component</td></tr><tr><td>Micro-drill (stereotaxic compatible)</td><td>Stoelting / Local supplier</td><td>&amp;mdash;</td><td>For craniotomy</td></tr><tr><td>Microinjection pump (e.g., Stoelting)</td><td>Stoelting/WPI</td><td>53210</td><td>Automated infusion pump for viral injections</td></tr><tr><td>Micropipette (P1000, P200, P10) and tips</td><td>-</td><td>-</td><td></td></tr><tr><td>Microscopy images (IHC sections)</td><td>Generated in-house</td><td>N/A</td><td>Immunohistochemistry images to be analyzed</td></tr><tr><td>Microtome blades (low-profile, disposable)</td><td>-</td><td>-</td><td></td></tr><tr><td>Midazolam (5.0 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Anesthetic component</td></tr><tr><td>Mounting medium</td><td>-</td><td>-</td><td></td></tr><tr><td>Naloxone (0.4 mg/mL)</td><td>Local supplier</td><td>&amp;mdash;</td><td>Antagonist of fentanyl</td></tr><tr><td>Needle holder and fine dissecting needles</td><td>-</td><td>-</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>-</td><td>-</td><td></td></tr><tr><td>O.C.T. compound</td><td>Tissue-Tek</td><td></td><td></td></tr><tr><td>Ophthalmic ointment (Bepanthen Eye and Nose Ointment, 5% Dexpanthenol)</td><td>Bayer</td><td>11568-0053</td><td>Prevents corneal drying during surgery</td></tr><tr><td>Orbital mixer</td><td>-</td><td>-</td><td></td></tr><tr><td>p24 ELISA kit</td><td>ZeptoMetrix</td><td>0801111</td><td></td></tr><tr><td>Parafilm</td><td>-</td><td>-</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>-</td><td>-</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>PBS without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;, pH 7.0</td><td>Gibco/Thermo Fisher</td><td>10-010-031</td><td></td></tr><tr><td>pCMV-dR8.2</td><td>Addgene</td><td>Plasmid #12263</td><td></td></tr><tr><td>Penicillin-Streptomycin (100&amp;times;)</td><td>Gibco/Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Pentobarbital</td><td>-</td><td>-</td><td></td></tr><tr><td>Phenylhydrazine</td><td>-</td><td>-</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>Plasmid #12259</td><td></td></tr><tr><td>Polyethylenimine (PEI), 1 mg/mL</td><td>Polysciences</td><td>23966</td><td></td></tr><tr><td>Primary antibody</td><td>-</td><td>-</td><td></td></tr><tr><td>pRSV-Rev</td><td>Addgene</td><td>Plasmid #12253</td><td></td></tr><tr><td>qPCR Lentivirus Titration Kit</td><td>abm</td><td>LV900</td><td></td></tr><tr><td>Smooth brush or fire-polished glass Pasteur pipettes (rounded tip)</td><td>-</td><td>-</td><td></td></tr><tr><td>Sodium azide</td><td>-</td><td>-</td><td></td></tr><tr><td>Spatula (brain lifter)</td><td>-</td><td>-</td><td></td></tr><tr><td>Stereotaxic frame (for mice)</td><td>Stoelting/WPI</td><td>51730</td><td>For accurate targeting of brain regions</td></tr><tr><td>Stericup 0.22 &amp;micro;m filter unit</td><td>MilliporeSigma</td><td>SCGPU02RE</td><td></td></tr><tr><td>Sucrose</td><td>-</td><td>-</td><td></td></tr><tr><td>Sutures (e.g., nylon 5-0)</td><td>Ethicon / Local supplier</td><td>1664G</td><td>For wound closure</td></tr><tr><td>T75 and T175 cell culture flasks</td><td>SPL Life Sciences</td><td>70075/71175</td><td></td></tr><tr><td>Transfer plasmid (gene of interest)</td><td>-</td><td>-</td><td></td></tr><tr><td>Triton X-100</td><td>-</td><td>-</td><td></td></tr><tr><td>Ultracentrifuge with JS-24.38 rotor</td><td>Beckman Coulter</td><td>JS-24.38</td><td></td></tr><tr><td>Vortex mixer</td><td>-</td><td>-</td><td></td></tr><tr><td>Water bath (37 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Xylene substitute</td><td>-</td><td>-</td><td></td></tr></tbody></table>

lentiviral-induced striatal pathology as a preclinical model of polyglutamine spinocerebellar ataxias

Lentiviral-Induced Striatal Pathology as a Preclinical Model of Polyglutamine Spinocerebellar Ataxias

Polyglutamine spinocerebellar ataxias are a group of six neurodegenerative diseases caused by a mutation in the trinucleotide CAG tract within the coding region of each causative gene. This mutation leads to an abnormally expanded polyglutamine stretch in the protein, making it prone to aggregation and leading to the formation of intraneuronal inclusions, which are a hallmark of these diseases. The molecular mechanisms underlying the pathogenesis of these diseases are complex and not fully understood. So far, these diseases remain incurable. Understanding molecular mechanisms in vulnerable brain regions is essential for therapeutic development. Flexible, region-specific experimental platforms are needed to rapidly test mechanisms and interventions without the time and cost of generating multiple transgenic lines, even though not addressing selective vulnerability. In recent years, we and others have developed several lentiviral-based models to study the impact of expanded protein expression in specific regions of the brain. These models show neuropathological and behavioral features that mimic some disease features, highlighting their importance in understanding the pathogenesis and in the development of new therapeutic strategies. In this paper, we describe the development of a striatal model using SCA2 as an example. We highlight lentiviral vector development and production, and the stereotaxic injection of the vectors expressing the expanded form of ataxin-2 (the disease-causing protein). Lastly, we characterize two main neuropathological hallmarks: the formation of inclusions and the loss of neuronal markers. By providing a controlled platform that reproduces these key pathological features, this model offers a valuable tool to dissect region-specific mechanisms of neurodegeneration and to evaluate targeted therapeutic interventions.

This study successfully identified the expression of ataxin-2 and DARPP-32 using the present protocol. Following stereotaxic injection of lentiviral vectors into the striatum of C57BL/6J mice, robust expression of both wild-type and mutant ataxin-2 was detected after 12 weeks. We showed previously12 that these constructs are specific to neurons, effectively transducing an average of 20,000 neurons per mm3. Under our experimental conditions, immunostaining for ataxin-2 revealed ...

Polyglutamine spinocerebellar ataxias are caused by CAG expansions producing aggregation-prone proteins. This protocol describes vector production, stereotaxic delivery, and analysis of inclusions and neuronal marker loss, providing a controlled platform to investigate mechanisms and evaluate targeted therapies for neurodegenerative diseases.

Polyglutamine spinocerebellar ataxias are a group of six neurodegenerative diseases caused by a mutation in the trinucleotide CAG tract within the coding ...

This study reports the development of a lintiviral mouse model for rare neurodegenerative diseases, specifically spinocerellar ataxias. This protocol develops mouse models with some advantages compared with conventional trangenic mouse models. Namely, they are faster, they are cost affordable, and they allow a specific transaction in specific brain regions.This is suitable for mechanistic studies and for the development of therapeutic strategies. To begin, position the anesthetized C57BL6J mouse on a clean desk and confirm the depth of anesthesia by assessing the toe pinch reflex. After shaving the animal scalp, place it in the stereotaxic frame on a heating pad and position the head using the ear bars and bite bar.Mount the syringe containing two microliters of lentiviral vector solution into the automated microinjection pump. Set the pump parameters, including a speed of 0.25 microliters per minute, a volume per hemisphere of two microliters, and the appropriate syringe type. Prepare the surgical site using alternating rounds of povidone iodine or chlorhexidine-based scrub and 70%alcohol repeated three times before making the incision.Using a sterile scalpel blade, make a midline incision of approximately one to 1.5 centimeters from between the ears to the eyes. Expose the skull and clean the surface by removing the periosteum with cotton swabs. Locate bregma and record the stereotaxic zero point manually or digitally.Determine the injection coordinates relative to bregma with anteroposterior at plus 0.6 millimeters and mediolateral at plus or minus 1.8 millimeters. Drill carefully at the marked anteroposterior and mediolateral coordinates slowly to avoid penetrating the brain. Lower the injection needle slowly to the dorsoventral coordinate, and then test for needle clogging by injecting a portion of the solution until a drop is clearly visible.Test that the hole is in the correct position by lowering the injection needle. Inject two microliters of lentivirus encoding for the wild-type ataxin into one hemisphere at a rate of 0.25 microliters per minute. After five minutes, slowly retract the needle to avoid reflux.Inject the mutant or expanded protein into the contralateral side similarly. Close the incision with three to five interrupted 5-O nylon sutures. Administer a mixture of antagonistic drugs subcutaneously to reverse the effects of the initial anesthetics before returning the animal to its recovery cage and isolate the brain after 12 weeks.Fill the wells of a 48 well plate with PBS containing 0.05%sodium azide. Then, transport the brain on dry ice and allow it to equilibrate inside the precooled cryostat for five to 10 minutes to prevent tissue damage. Using a scalpel, remove the olfactory bulbs as they are soft and easy to cut.Apply a pea size drop of fresh OCT compound onto the specimen disc and place the cerebellum in contact. Once frozen, add additional OCT to embed the entire brain. Mount the specimen disc in the holder and trim until the striatum visible as two white circles in each hemisphere is observed.If hemispheres received different treatments, pierce one hemisphere with a needle to maintain orientation and record this for later analysis. Next, switch to sectioning mode on the instrument panel and collect free floating sections in a 48-well plate using a fine brush or a fire polished glass pasture pipette. Place the first section in well A1 and the second in A2, continuing sequentially.When the plate is full, either continue in a new plate or proceed until the entire striatum is sectioned. Seal the plates with transparent film and store at four degrees Celsius until use. Perform immunohistochemistry on the sections using appropriate antibodies and develop the staining with diaminobenzadine.Monitor the development of the brown color and allow the reaction to reach the expected intensity level. Capture images of coronal brain sections spanning the entire striatum. For manual counting, open the image in Fiji to load it and calibrate the pixel size if required when metadata is not available.Select, analyze and choose set scale to enter the known distance, such as one pixel equals 0.5 micrometers. Navigate to plugins, then analyze cell counter and select cell counter. Click initialize and select the image.Select a marker type such as type one for aggregates. Manually click on each visible aggregate to label it. Record the count displayed in the cell counter panel for each image or section, and export the counts manually or compile results using a macro.For semi-automated quantification, open IHC Aggregount and click upload image to load the image. View the image in the original image display and navigate to the processing tab. Apply high-low to enhance aggregate and background contrast.Apply a brightness threshold to filter aggregates from the background and use the min-size filter to remove small particles. Finally, navigate to the general tab for immunohistochemistry aggregate detection. Click Calculate Total Aggregates and Calculate Aggregate Area.Observe detected aggregates highlighted and listed in the matrix table with identification, area, and mean size. Robust expression of both wild type and mutant ataxin-2 was detected 12 weeks after the stereotaxic injection of lentiviral vectors into the striatum of C57BL6J mice. Immunostaining for ataxin-2 revealed a diffuse cytoplasmic distribution of wild-type ataxin-2 without forming discrete inclusions.In contrast, the mutant ataxin-2 exhibited cytoplasmic aggregates with a distinct spherical morphology. Immunolabeling with DARP-32, a marker for medium spiny neurons, demonstrated that both wild-type and mutant ataxin-2 were expressed within the striatal neuronal population. While wild-type ataxin-2 expression did not alter the gross morphology of DARP-32 positive neurons, the mutant protein produced neuronal marker loss.This protocol allowed a segment of aggregation and neuronal loss within specific brain region, namely striatum, in response to therapeutic intervention. The main challenge of this protocol is to achieve precise lintiviral delivery via accurate stereotoxic surgery, controlled biodosage and minimizing tissue damage for consistent results. Future work can use this model to identify molecular mechanism underlying these pathogenesis and to accelerate the development of target therapy and biomarkers.

lentiviral-induced-striatal-pathology-as-preclinical-model

Research

JoVE Journal

Neuroscience

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

Source: Tagliafierro, L., et al. Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models. J. Vis. Exp. (2019)This video demonstrates the lentiviral transduction of neural progenitor cells (NPCs) for targeted epigenetic modification of alpha-synuclein, a key protein in Parkinson’s disease. The protocol enables methyltransferase-mediated DNA methylation, reducing SNCA expression for studying gene regulation...

Lentiviral Transduction of Neural Progenitor Cells for Epigenetic Regulation of Alpha-Synuclein Expression

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene...

Cerebellar Regional Dissection for Molecular Analysis

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson's disease. These vectors encoding the human α-synuclein are delivered into the substantia nigra to recapitulate the synuclein pathology associated with Parkinson's disease.

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Discover lentiviral-based striatal modeling for polyglutamine spinocerebellar ataxias using stereotaxic injection in C57BL6J mice. This method enables region-specific expression of expanded ataxin-2 to study disease mechanisms and evaluate therapeutic interventions.

Lentiviral-Induced Striatal Pathology as a Preclinical Model of Polyglutamine Spinocerebellar Ataxias

Explore More Videos