Arginine methylation of RNA-binding proteins is impaired in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD.

Mutant VPS35-D620N induces motor dysfunction and impairs DAT-mediated dopamine recycling pathway.

The D620N mutation in vacuolar protein sorting protein 35 (VPS35) gene has been identified to be linked to late onset familial Parkinson disease (PD). However, the pathophysiological roles of VPS35-D620N in PD remain unclear. Here, we generated the transgenic Caenorhabditis elegans overexpressing either human wild type or PD-linked mutant VPS35-D620N in neurons. C. elegans expressing VPS35-D620N, compared with non-transgenic controls, showed movement disorders and dopaminergic neuron loss. VPS35-D620N worms displayed more swimming induced paralysis but showed no defects in BSR assays, thus indicating the disruption of dopamine (DA) recycling back inside neurons. Moreover, VPS35 formed a protein interaction complex with DA transporter (DAT), RAB5, RAB11 and FAM21. In contrast, the VPS35-D620N mutant destabilized these interactions, thus disrupting DAT transport from early endosomes to recycling endosomes, and decreasing DAT at the cell surface. These effects together increased DA in synaptic clefts, and led to dopaminergic neuron degeneration and motor dysfunction. Treatment with reserpine significantly decreased the swimming induced paralysis in VPS35-D620N worms, as compared with vehicle treated VPS35-D620N worms. Our studies not only provide novel insights into the mechanisms of VPS35-D620N-induced dopaminergic neuron degeneration and motor dysfunction via disruption of DAT function and the DA signaling pathway but also indicate a potential strategy to treat VPS35-D620N-related PD and other disorders.

Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies.

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.

Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.

Mutant-TMEM230-induced neurodegeneration and impaired axonal mitochondrial transport.

Parkinson's disease (PD) is a neurodegenerative disease with movement disorders including resting tremor, rigidity, bradykinesia and postural instability. Recent studies have identified a new PD associated gene, TMEM230 (transmembrane protein 230). However, the pathological roles of TMEM230 and its variants are not fully understood. TMEM230 gene encodes two protein isoforms. Isoform2 is the major protein form (~95%) in human. In this study, we overexpress isoform2 TMEM230 variants (WT or PD-linked *184Wext*5 mutant) or knockdown endogenous protein in cultured SH-5Y5Y cells and mouse primary hippocampus neurons to study their pathological roles. We found that overexpression of WT and mutant TMEM230 or knockdown of endogenous TMEM230-induced neurodegeneration and impaired mitochondria transport at the retrograde direction in axons. Mutant TMEM230 caused more severe neurotoxicity and mitochondrial transport impairment than WT-TMEM230 did. Our results demonstrate that maintaining TMEM230 protein levels is critical for neuron survival and axon transport. These findings suggest that mutant-TMEM230-induced mitochondrial transport impairment could be the early event leading to neurite injury and neurodegeneration in PD development.

Immortalized striatal precursor neurons from Huntington's disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics.

We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.

Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases.

Significant progress has been made in understanding the pre-symptomatic phase of amyotrophic lateral sclerosis. While much is still unknown, advances in other neurodegenerative diseases offer valuable insights. Indeed, it is increasingly clear that the well-recognized clinical syndromes of Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy and frontotemporal dementia are also each preceded by a pre-symptomatic or prodromal period of varying duration, during which the underlying disease process unfolds, with associated compensatory changes and loss of inherent system redundancy. Key insights from these diseases highlight opportunities for discovery in amyotrophic lateral sclerosis. The development of biomarkers reflecting amyloid and tau has led to a shift in defining Alzheimer's disease based on inferred underlying histopathology. Parkinson's disease is unique among neurodegenerative diseases in the number and diversity of non-genetic biomarkers of pre-symptomatic disease, most notably REM sleep behaviour disorder. Huntington's disease benefits from an ability to predict the likely timing of clinically manifest disease based on age and CAG-repeat length alongside reliable neuroimaging markers of atrophy. Spinal muscular atrophy clinical trials have highlighted the transformational value of early therapeutic intervention, and studies in frontotemporal dementia illustrate the differential role of biomarkers based on genotype. Similar advances in amyotrophic lateral sclerosis would transform our understanding of key events in pathogenesis, thereby dramatically accelerating progress towards disease prevention. Deciphering the biology of pre-symptomatic amyotrophic lateral sclerosis relies on a clear conceptual framework for defining the earliest stages of disease. Clinically manifest amyotrophic lateral sclerosis may emerge abruptly, especially among those who harbour genetic mutations associated with rapidly progressive amyotrophic lateral sclerosis. However, the disease may also evolve more gradually, revealing a prodromal period of mild motor impairment preceding phenoconversion to clinically manifest disease. Similarly, cognitive and behavioural impairment, when present, may emerge gradually, evolving through a prodromal period of mild cognitive impairment or mild behavioural impairment before progression to amyotrophic lateral sclerosis. Biomarkers are critically important to studying pre-symptomatic amyotrophic lateral sclerosis and essential to efforts to intervene therapeutically before clinically manifest disease emerges. The use of non-genetic biomarkers, however, presents challenges related to counselling, informed consent, communication of results and limited protections afforded by existing legislation. Experiences from pre-symptomatic genetic testing and counselling, and the legal protections against discrimination based on genetic data, may serve as a guide. Building on what we have learned-more broadly from other pre-symptomatic neurodegenerative diseases and specifically from amyotrophic lateral sclerosis gene mutation carriers-we present a road map to early intervention, and perhaps even disease prevention, for all forms of amyotrophic lateral sclerosis.

G2019S-LRRK2 mutation enhances MPTP-linked Parkinsonism in mice.

Parkinson's disease (PD) is a common neurodegenerative disease with a heterogeneous etiology that involves genetic and environmental factors or exogenous. Current LRRK2 PD animal models only partly reproduce the characteristics of the disease with very subtle dopaminergic neuron degeneration. We developed a new model of PD that combines a sub-toxic MPTP insult to the G2019S-LRRK2 mutation. Our newly generated mice, overexpressing mutant G2019S-LRRK2 protein in the brain, displayed a mild, age-dependent progressive motor impairment, but no reduction of lifespan. Cortical neurons from G2019S-LRRK2 mice showed an increased vulnerability to stress insults, compared with neurons overexpressing wild-type WT-LRRK2, or non-transgenic (nTg) neurons. The exposure of LRRK2 transgenic mice to a sub-toxic dose of MPTP resulted in severe motor impairment, selective loss of dopamine neurons and increased astrocyte activation, whereas nTg mice with MPTP exposure showed no deficits. Interestingly, mice overexpressing WT-LRRK2 showed a significant impairment that was milder than for the mutant G2019S-LRRK2 mice. L-DOPA treatments could partially improve the movement impairments but did not protect the dopamine neuron loss. In contrast, treatments with an LRRK2 kinase inhibitor significantly reduced the dopaminergic neuron degeneration in this interaction model. Our studies provide a novel LRRK2 gene-MPTP interaction PD mouse model, and a useful tool for future studies of PD pathogenesis and therapeutic intervention.

Nemo-like kinase reduces mutant huntingtin levels and mitigates Huntington's disease.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, is highly expressed in the brain, but its function in the adult brain remains not well understood. In this study, we identify NLK as an interactor of huntingtin protein (HTT). We report that NLK levels are significantly decreased in HD human brain and HD models. Importantly, overexpression of NLK in the striatum attenuates brain atrophy, preserves striatal DARPP32 levels and reduces mutant HTT (mHTT) aggregation in HD mice. In contrast, genetic reduction of NLK exacerbates brain atrophy and loss of DARPP32 in HD mice. Moreover, we demonstrate that NLK lowers mHTT levels in a kinase activity-dependent manner, while having no significant effect on normal HTT protein levels in mouse striatal cells, human cells and HD mouse models. The NLK-mediated lowering of mHTT is associated with enhanced phosphorylation of mHTT. Phosphorylation defective mutation of serine at amino acid 120 (S120) abolishes the mHTT-lowering effect of NLK, suggesting that S120 phosphorylation is an important step in the NLK-mediated lowering of mHTT. A further mechanistic study suggests that NLK promotes mHTT ubiquitination and degradation via the proteasome pathway. Taken together, our results indicate a protective role of NLK in HD and reveal a new molecular target to reduce mHTT levels.

SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F.

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG-initiated polyGln and a polyAla protein expressed by repeat-associated non-ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady-state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.

Huntingtin silencing delays onset and slows progression of Huntington's disease: a biomarker study.

Huntington's disease is a dominantly inherited, fatal neurodegenerative disorder caused by a CAG expansion in the huntingtin (HTT) gene, coding for pathological mutant HTT protein (mHTT). Because of its gain-of-function mechanism and monogenic aetiology, strategies to lower HTT are being actively investigated as disease-modifying therapies. Most approaches are currently targeted at the manifest stage, where clinical outcomes are used to evaluate the effectiveness of therapy. However, as almost 50% of striatal volume has been lost at the time of onset of clinical manifest, it would be preferable to begin therapy in the premanifest period. An unmet challenge is how to evaluate therapeutic efficacy before the presence of clinical symptoms as outcome measures. To address this, we aim to develop non-invasive sensitive biomarkers that provide insight into therapeutic efficacy in the premanifest stage of Huntington's disease. In this study, we mapped the temporal trajectories of arteriolar cerebral blood volumes (CBVa) using inflow-based vascular-space-occupancy (iVASO) MRI in the heterozygous zQ175 mice, a full-length mHTT expressing and slowly progressing model with a premanifest period as in human Huntington's disease. Significantly elevated CBVa was evident in premanifest zQ175 mice prior to motor deficits and striatal atrophy, recapitulating altered CBVa in human premanifest Huntington's disease. CRISPR/Cas9-mediated non-allele-specific HTT silencing in striatal neurons restored altered CBVa in premanifest zQ175 mice, delayed onset of striatal atrophy, and slowed the progression of motor phenotype and brain pathology. This study-for the first time-shows that a non-invasive functional MRI measure detects therapeutic efficacy in the premanifest stage and demonstrates long-term benefits of a non-allele-selective HTT silencing treatment introduced in the premanifest Huntington's disease.

Physiological and pathological roles of LRRK2 in the nuclear envelope integrity.

Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson's disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 -/- mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.

RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

N6-Furfuryladenine is protective in Huntington's disease models by signaling huntingtin phosphorylation.

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.

GTP-binding inhibitors increase LRRK2-linked ubiquitination and Lewy body-like inclusions.

Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.

Synaptic dysregulation in a human iPS cell model of mental disorders.

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders, and 'a disease of synapses' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies, little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare, multiply affected, large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and, furthermore, dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders.

Genetic disruption of ankyrin-G in adult mouse forebrain causes cortical synapse alteration and behavior reminiscent of bipolar disorder.

Genome-wide association studies have implicated the ANK3 locus in bipolar disorder, a major human psychotic illness. ANK3 encodes ankyrin-G, which organizes the neuronal axon initial segment (AIS). We generated a mouse model with conditional disruption of ANK3 in pyramidal neurons of the adult forebrain (Ank-G cKO). This resulted in the expected loss of pyramidal neuron AIS voltage-gated sodium and potassium channels. There was also dramatic loss of markers of afferent GABAergic cartridge synapses, resembling the cortical microcircuitry changes in brains from psychotic patients, and suggesting disinhibition. Expression of c-fos was increased in cortical pyramidal neurons, consistent with increased neuronal activity due to disinhibition. The mice showed robust behavioral phenotypes reminiscent of aspects of human mania, ameliorated by antimania drugs lithium and valproate. Repeated social defeat stress resulted in repeated episodes of dramatic behavioral changes from hyperactivity to "depression-like" behavior, suggestive of some aspects of human bipolar disorder. Overall, we suggest that this Ank-G cKO mouse model recapitulates some of the core features of human bipolar disorder and indicates that cortical microcircuitry alterations during adulthood may be involved in pathogenesis. The model may be useful for studying disease pathophysiology and for developing experimental therapeutics.

Mutant G2019S-LRRK2 Induces Abnormalities in Arteriolar Cerebral Blood Volume in Mouse Brains: An MRI Study.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease and the most common movement disorder characterized by motor impairments resulting from midbrain dopamine neuron loss. Abnormalities in small pial arteries and arterioles, which are the primary pathways of local delivery of nutrients and oxygen in brain tissue, have been reported in many neurodegenerative diseases including PD. Mutations in LRRK2 cause genetic PD and contribute to sporadic PD. The most common PD-linked mutation LRRK2 G2019S contributes 20-47% of genetic forms of PD in Caucasian populations. The human LRRK2 G2019S transgenic mouse model displays PD-like movement impairment and was used to identify novel LRRK2 inhibitors, which provides a useful model for studying microvascular abnormalities in PD. OBJECTIVES: To investigate abnormalities in arteriolar cerebral blood volume (CBVa) in various brain regions using the inflow-based vascular-space occupancy (iVASO) MRI technique in LRRK2 mouse models of PD. METHODS: Anatomical and iVASO MRI scans were performed in 5 female and 7 male nontransgenic (nTg), 3 female and 4 male wild-type (WT) LRRK2, and 5 female and 7 male G2019S-LRRK2 mice of 9 months of age. CBVa was calculated and compared in the substantia nigra (SN), olfactory cortex, and prefrontal cortex. RESULTS: Compared to nTg mice, G2019S-LRRK2 mice showed decreased CBVa in the SN, but increased CBVa in the olfactory and prefrontal cortex in both male and female groups, whereas WT-LRRK2 mice showed no change in CBVa in the SN (male and female), the olfactory (female), and prefrontal (female) cortex, but a slight increase in CBVa in the olfactory and prefrontal cortex in the male group only. CONCLUSIONS: Alterations in the blood volume of small arteries and arterioles (CBVa) were detected in the G2019S-LRRK2 mouse model of PD. The opposite changes in CBVa in the SN and the cortex indicate that PD pathology may have differential effects in different brain regions. Our results suggest the potential value of CBVa as a marker for clinical PD studies.

Pridopidine protects neurons from mutant-huntingtin toxicity via the sigma-1 receptor.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin gene (HTT), translated into a Huntingtin protein with a polyglutamine expansion. There is preferential loss of medium spiny neurons within the striatum and cortical pyramidal neurons. Pridopidine is a small molecule showing therapeutic potential in HD preclinical and clinical studies. Pridopidine has nanomolar affinity to the sigma-1 receptor (sigma-1R), which is located predominantly at the endoplasmic reticulum (ER) and mitochondrial associated ER membrane, and activates neuroprotective pathways. Here we evaluate the neuroprotective effects of pridopidine against mutant Huntingtin toxicity in mouse and human derived in vitro cell models. We also investigate the involvement of the sigma-1 receptor in the mechanism of pridopidine. Pridopidine protects mutant Huntingtin transfected mouse primary striatal and cortical neurons, with an EC50 in the mid nanomolar range, as well as HD patient-derived induced pluripotent stem cells (iPSCs). This protection by pridopidine is blocked by NE-100, a purported sigma-1 receptor antagonist, and not blocked by ANA-12, a reported TrkB receptor antagonist. 3PPP, a documented sigma-1 receptor agonist, shows similar neuroprotective effects. Genetic knock out of the sigma-1 receptor dramatically decreases protection from pridopidine and 3PPP, but not protection via brain derived neurotrophic factor (BDNF). The neuroprotection afforded by pridopidine in our HD cell models is robust and sigma-1 receptor dependent. These studies support the further development of pridopidine, and other sigma-1 receptor agonists as neuroprotective agents for HD and perhaps for other disorders.

Regional subcortical shape analysis in premanifest Huntington's disease.

Huntington's disease (HD) involves preferential and progressive degeneration of striatum and other subcortical regions as well as regional cortical atrophy. It is caused by a CAG repeat expansion in the Huntingtin gene, and the longer the expansion the earlier the age of onset. Atrophy begins prior to manifest clinical signs and symptoms, and brain atrophy in premanifest expansion carriers can be studied. We employed a diffeomorphometric pipeline to contrast subcortical structures' morphological properties in a control group with three disease groups representing different phases of premanifest HD (far, intermediate, and near to onset) as defined by the length of the CAG expansion and the participant's age (CAG-Age-Product). A total of 1,428 magnetic resonance image scans from 694 participants from the PREDICT-HD cohort were used. We found significant region-specific atrophies in all subcortical structures studied, with the estimated abnormality onset time varying from structure to structure. Heterogeneous shape abnormalities of caudate nuclei were present in premanifest HD participants estimated furthest from onset and putaminal shape abnormalities were present in participants intermediate to onset. Thalamic, hippocampal, and amygdalar shape abnormalities were present in participants nearest to onset. We assessed whether the estimated progression of subcortical pathology in premanifest HD tracked specific pathways. This is plausible for changes in basal ganglia circuits but probably not for changes in hippocampus and amygdala. The regional shape analyses conducted in this study provide useful insights into the effects of HD pathology in subcortical structures.