Neurobiology of Disease

BackgroundDeep brain stimulation of the fornix (DBS-f) has emerged as a potential strategy for Alzheimers disease (AD), based on evidence that stimulating the Papez circuit may enhance hippocampal metabolism, increase hippocampal volume, and modulate large-scale memory networks. However, studies vary substantially in design, stimulation parameters, sample size, and disease stage, and the consistency of cognitive and motor effects remains unclear. ObjectiveTo systematically review and meta-analyze the cognitive outcome associated with fornix-targeted DBS in Alzheimers disease. MethodsThis systematic review followed PRISMA-2020 guidelines, and the protocol was registered in PROSPERO (ID: CRD420251175724). A comprehensive search was conducted in PubMed, Scopus, Embase, Web of Science (WOS) and Cochrane Database of Systematic reviews (CDSR) included randomized controlled trials (RCTs), cohort studies,and case series. Ten eligible studies were analyzed, including mild, moderate, and severe AD cohorts. Meta-analyses were performed for ADAS-Cog and MMSE outcomes using random-effects models. Subgroup analysis (observational vs RCT), trim-and-fill publication bias, sensitivity analysis, and meta-regression (age 63-65 years) were conducted. Structural and metabolic data (hippocampal volumetry and FDG-PET) and motor outcomes (FIM, ADL, Barthel Index) were narratively synthesized. ResultsAcross included studies, cognitive outcomes showed highly variable short-term responses but no sustained improvement in controlled settings. Severe-AD cohorts demonstrated early gains, MMSE and MoCA improved in 1.5-3 months (Mao 2018), and dual-target fornix + NBM DBS yielded significantly higher MMSE at 3 months (p=0.002) and MoCA at 3 (p=0.003) and 12 months (p=0.010) (Xu 2024). In contrast, mild-AD RCTs showed no clinically meaningful benefit. Meta-analysis demonstrated a null pooled effect for cognition: ADAS-Cog: SMD = 0.05 (95% CI spanning null), robust to trim-and-fill (k =1). MMSE: pooled SMD near zero, stable on leave-one-out sensitivity analysis. Subgroup comparisons (RCT vs observational) showed no differences ({chi}{superscript 2}=0.04-0.05, p>0.80), and meta-regression revealed no association between effect size and age ({beta} = -0.04, p=0.33; {tau}{superscript 2}=0), confirming minimal between-study variance. Structural and metabolic findings consistently showed biological activation despite weak clinical response. Two patients demonstrated bilateral hippocampal volume increase at 12 months, and FDG-PET studies reported widespread metabolic increases across frontal-temporal-parietal-striatal-thalamic and frontal-temporal-parietal-occipital-hippocampal networks. Higher baseline metabolism and increased metabolism at one year correlated with less cognitive decline. Motor outcomes showed no sustained improvements. FIM scores improved significantly more in the DBS group at 3 months (p<0.05) but not at 12 months (p=0.968). ADL and Barthel scores showed mixed responses in small severe-AD samples. DBS parameters were heterogeneous (1-7 V; 60-210 s; 130 Hz in most studies), and programming duration varied markedly, underscoring a lack of standardized neuromodulatory protocol. ConclusionFornix DBS reliably activates limbic and memory-related circuits at a physiological level but does not provide consistent or sustained cognitive or motor benefits at currently used parameters. Evidence suggests age- and severity-dependent effects, with older or more advanced patients showing transient improvements, while mild AD patients do not benefit. Future research should prioritize precision targeting, biomarker-driven patient selection, optimized stimulation paradigms, and multi-target neuromodulation approaches.

Essential tremor (ET), the most common movement disorder in adults, presents with involuntary shaking of the arms during postural hold and kinetic tasks linked to dysfunction in the cerebello-thalamo-cortical (CTC) network. Recently, transcutaneous afferent patterned stimulation (TAPS), applied through a wrist-worn device, has emerged as a non-invasive therapy for medication refractory ET. However, its mechanism remains unclear. We hypothesize that TAPS reduces tremor through modulation of the VIM thalamus in the CTC network. Employing refractory ET patients seeking VIM deep brain stimulation (DBS), we quantified clinical tremor improvement following TAPS treatment in a pre-operative setting, followed by intra-operative, microelectrode recording of the contralateral thalamus with concurrent TAPS treatment on and off. After one preoperative session, TAPS significantly reduces upper limb tremor, with asymmetric effect favoring the treated limb and greatest improvement tending to kinetic tremor. The magnitude of TAPS-related tremor reduction demonstrates a positive correlation with the modulation of alpha and beta band LFPs in the VIM. TAPS also modulated spiking activity in the VIM, though it was uncorrelated with the degree of tremor reduction. Of note, TAPS related modulation of LFPs and spiking activity was greatest near the optimal placement location for DBS lead in treating ET. In sum, TAPS likely reduces tremor in ET by modulating the VIM and connected nodes in the cerebello-thalamo-cortical pathway.

Oligodendrocyte dysfunction, myelin degeneration, and white matter structural alterations are critical events in Alzheimers disease (AD) that contribute to cognitive decline. A key hallmark of AD, A{beta} oligomers, disrupt oligodendrocyte and myelin homeostasis, but a comprehensive global analysis of the mechanisms involved is lacking. Here, transcriptomic profiling of A{beta}-exposed oligodendrocytes revealed widespread gene expression changes, particularly affecting pathways related to RNA localisation. Among the genes identified, we focused on Hnrnpa2/b1, the gene encoding the hnRNP A2 protein, which is essential for RNA transport and translation of myelin proteins. We confirmed aberrant upregulation of hnRNP A2 in hippocampal oligodendrocytes from post-mortem human brains of early-stage AD patients, A{beta}-injected mouse hippocampi and A{beta}-treated disrupting cells in vitro. RIP-seq analysis of the hnRNP A2 interactome revealed attenuated interactions with Hnrnpk and Hnrnpa2/b1, while interactions with Mbp and Mobp were enriched, suggesting changes in RNA metabolism of molecules associated with mRNA transport of myelin proteins. A{beta} increased the total number and dynamics of mRNA-containing granules, facilitating local translation of the myelin proteins MBP and MOBP and attenuating Ca2+ signalling. These findings suggest that A{beta} oligomers disrupt RNA metabolism mechanisms crucial for oligodendrocyte myelination through dysregulation of hnRNP A2 and myelin protein levels, potentially affecting oligodendroglia Ca2+ homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/590214v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@19e046eorg.highwire.dtl.DTLVardef@134f15corg.highwire.dtl.DTLVardef@d1f8aaorg.highwire.dtl.DTLVardef@11c97af_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

Idiopathic/isolated REM sleep behavior disorder (iRBD) is considered a prodromal stage of alpha-synucleinopathies. Cortical and sub-cortical brain modifications begin years before the emergence of overt neurodegenerative symptoms. To better understand the pathophysiological process impacting the brain from the prodromal to the overt stage of alpha-synucleinopathy, it is essential to assess iRBD patients over time. Recent evidence suggests that the human brain operates at an operating point near a critical phase transition between subcritical and supercritical phases in the systems state space to maintain cognitive and physiological performance. In contrast, a deviation from the critical regime leading to altered oscillatory dynamics has been observed in several pathologies. Here, we investigated if the alpha-synucleinopathy produces a deviation of the operating point already evident in the prodromal phase and if this shift correlates with biological and clinical disease severity. We analyzed a dataset of 59 patients with iRBD (age 69.61 {+/-} 6.98, 50 male) undergoing resting-state high-density EEG, presynaptic dopaminergic imaging, and clinical evaluations. Thirty-one patients (age 72.41 {+/-} 7.05, 31 male) also underwent clinical and instrumental follow-up (mean follow-up period 25.85 {+/-} 10.20 months). To localize the individual operating points along the excitation-inhibition (EI) continuum, we assessed both measures of neuronal EI balance and measures of critical brain dynamics such as long-range temporal correlation (LRTCs) and neuronal bistability in spontaneous narrow-band oscillations. Finally, we correlated critical brain dynamics and EI balance metrics with phase synchronization, nigro-striatal dopaminergic functioning, and clinical performances. Compared to 48 healthy subjects (age 70.25 {+/-} 10.15, 23 male), iRBD patients showed higher values of LRTCs and bistability in the 2-7 Hz band at diagnosis. Patients who eventually phenoconverted to overt alpha-synucleinopathy exhibited a more excitation-dominated (fEI > 1) condition than stable iRBD patients in 5-7 Hz. This higher excitation also directly correlated with phase synchronization in 2-7 Hz, further suggesting a shift of the operating point toward a supercritical state with the disease progression. Moreover, excitation-dominated state and low bistability were associated with deterioration of the nigro-striatal dopaminergic function and tended to correlate with stronger clinical symptoms. In conclusion, this study shows for the first time a deviation of the working point from inhibition-to excitation-dominated states along the continuum from prodromal to overt phases of the disease. These cortical brain dynamics modifications are associated with nigro-striatal dopaminergic impairment. These results increase our knowledge of the physiopathological process underlying alpha-synucleinopathies since prodromal stages, possibly providing new clues on disease-modifying strategies.

BackgroundSpinocerebellar ataxia type 2 (SCA2) is a polyglutamine disorder, and variants in its disease protein Ataxin-2 act as modifiers in the progression of Amyotrophic Lateral Sclerosis. There are no reliable molecular progression biomarkers for SCA2. ObjectivesThe aim of this study was to define novel molecular progression biomarker candidates for SCA2. MethodsUsing cerebellar and cervicothoracic spinal cord RNA from Atxn2-CAG100-KnockIn and wildtype mice, a multi-omics study was conducted, followed by validation in mice and humans. Global transcriptome studies were conducted using the Clariom D microarray. Extracted proteins were analyzed by LC-MS/MS for global proteomics, and Immobilized Metal Affinity Chromatography for phosphoproteomics. Validation assessed expression by RT-qPCR, and protein abundance by quantitative immunoblots and ELISA. Patients with SCA2 were diagnosed following standard procedures, and the age at onset, SARA score, INAS count, and disease duration were used as clinical severity markers. ResultsVenn diagram comparisons across all OMICS datasets indicated that only Serpinb1a-transcript, SERPINB1A-protein and -phosphopeptides were consistently downregulated at terminal stage in 14-month-old KnockIn mice. Expression studies in cerebellum and spinal cord from 10 weeks (pre-manifest), 6-month-old (early ataxic), and 14-month-old (late ataxic stage) mice confirmed this progressive decrease at mRNA and protein level. SERPINB1 plasma levels were significantly lower in SCA2 patients, and displayed a significant association with the CAG repeat length at expanded ATXN2 alleles and the age at onset, also showing a trend towards significance with the SARA score. ConclusionsSERPINB1 was identified as novel promising biomarker with specificity for SCA2 pathomechanisms.

Huntingtons disease (HD) is caused by polyglutamine (polyQ) expansions in huntingtin (HTT). Polyglutamine repeat lengths >35Q lead to neurodegeneration and longer repeats correspond to earlier symptom onset. HTT scaffolds kinesin-1 and dynein to a variety of vesicles and organelles directly and through adaptors. To characterize the effects of HTT polyQ expansions on axonal transport, we tracked BDNF vesicles, mitochondria, and lysosomes in neurons induced from an isogenic set of human stem cell lines with repeat lengths of 30, 45, 65, and 81Q. Mild and intermediate pathogenic polyQ expansions caused increased BDNF motility, while HTT-81Q misdirected BDNF towards the distal tip. In comparison, mitochondria and lysosome transport showed mild defects with polyQHTT. We next examined the effect of polyQHTT in combination with neuroinflammatory stress. Under stress, BDNF cargoes in HTT-30Q neurons were more processive. Stress in HTT-81Q resulted in a stark decrease in the number of BDNF cargoes. However, the few remaining BDNF cargoes displayed more frequent long-range motility in both directions. Under neuroinflammatory stress, lysosomes were more abundant in HTT-81Q neurons, and motile lysosomes moved less processively and had an anterograde bias while lysosomes in HTT-30Q where not strongly affected. To examine how HTT-polyQ expansions altered the motors and adaptors on vesicular cargoes, we isolated BDNF cargoes from neurons and quantified the proteins associated with them. BDNF-endosomes isolated from HTT-81Q neurons associated with 2.5 kinesin-1 and 3.9 HAP1 molecules on average, compared to 1.0 kinesin-1 and 1.0 HAP1 molecule for HTT-30Q neurons. Together, these results show that polyQ expansions in HTT cause aberrant motor and adaptor recruitment to cargoes, resulting in dysregulated transport and responses to neuroinflammatory stress.

Amyloidogenic mutant huntingtin exon-1 (mHTTex1) protein aggregates with pathogenic polyglutamine (polyQ) tracts are the potential root cause of Huntingtons disease (HD). Here, we assessed the gain-of-function toxicity of mHTTex1 aggregation in neurons of HD transgenic flies. We show that the rate of mHTTex1 aggregation in neurons and early mortality of HD transgenic flies are correlated. We observed sequestration of key synaptic proteins into amyloid-like mHTTex1 aggregates and a concomitant decrease of their transcript levels, suggesting that progressive mHTTex1 aggregate stress in neurons leads to an impairment of synaptic function. Machine learning-based data analysis revealed that the abundance of synaptic proteins such as the vesicular monoamine transporter Vmat in the brain is predictive of fly survival. RNAi knockdown of Vmat-encoding transcripts in neurons with pathogenic amyloid-like HTTex1Q97 aggregates further shortened the lifespan of HD flies, supporting the hypothesis that mHTTex1 aggregation drives impairment of synaptic processes and pathogenesis of HD.

The cellular form of the prion protein (PrPC) is known for its involvement in the pathogenesis of prion diseases. Recent research implicates the physiological isoform of PrP in neuronal development, excitability, and synaptic plasticity, as well as in other biological processes. However, its precise function in the development and function of neurons remains poorly understood. Here, we investigated its role during different developmental stages, both in vitro and in vivo, using different PrP knock-out (KO) mouse lines (Prnp-/-). Prion protein KO neurons cultured on microelectrode arrays (MEAs) displayed altered network dynamics compared to wild type cultures, comprising reduced burst frequency, and abnormal spike patterns, indicative of impaired maturation of the synaptic circuitry. These functional alterations were associated with a reduced expression of key presynaptic and postsynaptic proteins, including elements of the SNARE complex and regulators of excitation-inhibition balance. Similar molecular changes were also confirmed in a second Prnp-/- model, suggesting that PrPC is directly involved in these mechanisms regardless of genetic backgrounds. Alterations in neuronal networks were traceable into adulthood: in vivo recordings in adult Prnp-/- mice revealed increased neuronal responses to visual danger stimuli, which correlated with behaviorally increased fear responses to those stimuli. Together, our findings support a critical role for PrPC in the establishment and maintenance of functional neuronal networks, from early developmental stages in vitro to behaviorally mature relevant circuits in vivo, beyond genomic background. These results indicate that PrPC acts as a key regulator of synaptic development and function both in physiological and pathological conditions.

Alzheimers disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined induced pluripotent stem cell (iPSC)-derived NGN2 induced neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding the disease pathogenesis.

In Gaucher and Niemann-Pick C diseases, the glucosylceramide (GlcCer) depletion hypothesis states that depletion of non-lysosomal sphingolipid pools can lead to dysfunction in the secretory and lysosomal system. The hypothesis suggests: 1) lysosomal dysfunction can be separated from lysosomal storage, 2) Lysosomal/secretory dysfunction/vATPase activity is corrected by increasing non-lysosomal GlcCer pools, and 3) Changes in higher glycosphingolipid synthesis due to changes in Golgi pH and/or GlcCer non-vesicular transport. Evidence for this mechanism includes 1) Successful treatment of cells and animals by imino sugar inhibition of the non-lysosomal neutral pH GlcCer hydrolase GBA2, 2) Increasing ER/cytosol GlcCer increases in vATPase regulatory V0a1 subunit expression. Heterozygous mutations in GBA1, a lysosomal glucocerebrosidase (GCase), cause GCase misfolding and mislocalisation in the ER/cytoplasm which is linked to Parkinsons disease (GBA-PD). Unexpectedly, similar to previous results in storing fibroblasts, N370S and L444P fibroblasts revealed increased endolysosomal pH and size despite the absence of glucolipid storage. Induction of storage by reducing residual lysosomal GCase activity in the N370S/L444P fibroblasts by the addition conduritol B-epoxide had no further effect on lysosomal function. In contrast, the addition of a soluble GlcCer analogue (adaGlcCer) reverses increased endolysosomal pH and volume in N370S mutant fibroblasts. The results are consistent with ER/cytosolic glucolipid depletion in GBA-PD fibroblasts. We discuss the potential for toxic/ectopic GBA1 hydrolysis and disrupted vATPase activity may lead to defective dopamine packaging and synaptic vesicle endocytosis as a new hypothesis in GBA-PD.

Personality changes following neurosurgical procedures pose a major concern for patients and remain poorly understood both by clinicians and neuroscientists. Here we report a case of a female patient in her 50s who underwent resection of a large sagittal sinus meningioma with bilateral extension, including resection and ligation of the superior sagittal sinus, that resulted in borderline personality disorder and symptoms resembling the Gastaut-Geschwind syndrome. Clinical observations were further reflected and experimentally quantified with a series of behavioral and neuroimaging tasks assessing self-other voice discrimination, one of the established markers for self-consciousness. In all tasks, the patient consistently confused self- and other voices - i.e., she misattributed other-voice stimuli to herself and self-voice stimuli to others. Moreover, behavioral findings were corroborated with scalp EEG results. Specifically, the same EEG microstate, that was in healthy participants associated with hearing their own voice, in this patient occurred more often for other-voice stimuli. We hypothesize that the patients preexisting psychological problems were significantly aggravated by postoperative decompensation of a fragile steady-state combination of direct frontal lobe compression and preoperative development of a large venous collateral hemodynamic network that followed gradual occlusion of the superior sagittal sinus. Resection of the sagittal sinus together with the tumor impacted venous drainage of brain areas associated with self-consciousness. These findings are of high relevance for developing experimental biomarkers of post-surgical personality alterations.

Genetic defects in AP2M1, which encodes the -subunit of the adaptor protein complex 2 (AP-2) essential for clathrin-mediated endocytosis (CME), cause a rare form of developmental and epileptic encephalopathy (DEE). In this study, we modeled AP2M1-DEE in Drosophila melanogaster to gain deeper insights into the underlying disease mechanisms. Pan-neuronal knock-down of the Drosophila AP2M1 ortholog, AP-2{micro}, resulted in a consistent heat-sensitive paralysis phenotype and altered morphology in class IV dendritic arborization (c4da) neurons. Unexpectedly, affected flies were resistant to antiseizure medications and exhibited increased resistance to electrically induced seizures. A CRISPR-engineered fly line carrying the recurrent human disease variant p.Arg170Trp displayed a milder seizure resistance phenotype. While these findings contrast with the human phenotype, they align with previous studies on other CME-related genes in Drosophila. Our results suggest that hyperexcitability and seizures in AP2M1-DEE may stem from broader defects in neuronal development rather than direct synaptic dysfunction.

Eye muscles and the motor neurons in the innervating cranial nerve nuclei are relatively spared in human ALS, and likewise, these cranial motor neurons are spared of SOD1YFP aggregation in a transgenic mouse model of SOD1-linked ALS, G85R SOD1YFP. RNA profiling of mouse oculomotor (CN3) neurons (resistant) vs hypoglossal (CN12) and spinal cord motor neurons (susceptible) from nontransgenic mice identified differentially expressed channel and receptor genes. A number were evaluated for effects on survival of the ALS strain by transgenesis or knockout to emulate the relative RNA level in oculomotor neurons. Transgenesis of Thy1.2-driven cDNA for mouse Kcnn1, a potassium channel subunit, extended the median days of survival time to paralysis of mutant G85R SOD1YFP mice by up to 100%, associated with absence of fluorescent aggregates; extended the median time to paralysis of G93A SOD1 mice by up to 55%; and extended the median time to endstage motor disease of a Thy1.2-driven alpha-synuclein transgenic strain by up to greater than 100%. The overexpressed Kcnn1 subunit was diffusely cytoplasmic in motor neurons and found to induce a multifaceted stress response as judged by RNAseq and immunostaining, including ER stress response, mitochondrial stress response, and an integrated stress response. Like other potassium channel subunits, Kcnn1 subunit is likely targeted to the ER, but as reported earlier in rodent Kcnn1-transfected cultured cells, in the absence of Kcnn2 with which to co-assemble, Kcnn1 is channel-inactive and is diffusely cytoplasmic. Thus, a nonassembled and potentially misfolded state of overexpressed Kcnn1 targeted to the ER of neurons may explain the stress responses, which in the mutant SOD1 and A53T alpha-synuclein mice, protect against the pathogenic proteins. Major neurodegenerative diseases, including Alzheimers Disease and Parkinsons Disease, are associated with the accumulation of characteristic proteins, Abeta/Tau and alpha-synuclein in AD and PD, respectively, that misfold, aggregate, and in many cases form amyloid fibrils (e.g. Long and Holtzman, 2019; Sierksma et al, 2020; Tanner et al, 2024). Such pathogenic behavior is associated with malfunction/death of specific neuronal populations, producing consequent clinical symptoms. It seems counterintuitive to observe proteinopathy as a major facet of these diseases considering that there is generally a quality control machinery in all cells, consisting of effectors - molecular chaperones, ubiquitin/proteasomal components, and autophagy/lysosome components - governed by a "sensor" circuitry - e.g. UPR, ISR, HSF - that can detect such misbehavior and induce protective responses. While neurons may be particularly susceptible because they are postmitotic and unable to distribute damaging protein species to daughter cells as a protective means, it has remained unclear whether the endogenous sensor/effector pathways can be induced sufficiently in vivo so as to mediate protection. Here, we report that neuronal overexpression of a potassium channel subunit, mouse Kcnn1, in two different transgenic mouse neurodegenerative models, protects against aggregation and cell loss by apparent induction of multiple stress response pathways, substantially extending survival of the mice.

A growing body of work has recently linked ventral tegmental area (VTA) dopamine neuron dysfunction to Alzheimers disease (AD). Work in AD mouse models suggests that VTA dopamine neurons are intrinsically hyperexcitable, yet release less dopamine and exhibit disrupted downstream signaling. Significant focus has been placed on describing dopamine release in projection regions; however, dopamine neurons somatodendritically integrate vast synaptic input, altering action potential output and ultimately determining neurotransmitter release. Synaptic transmission is broadly disrupted in AD, but it is not known to what extent excitatory and inhibitory inputs to the VTA are altered. Here we describe enhanced synaptic excitation in dopamine neurons in the amyloid + tau-driven 3xTg-AD mouse model. Patch-clamp electrophysiology experiments revealed enhanced AMPAR-mediated excitatory input in a subset of perisomatic connections. In contrast, GABAAR-mediated inhibition was decreased as a function of dendritic atrophy, determined by single neuron reconstructions. We also detected elevated protein kinase C (PKC) substrate levels in the midbrain, and pharmacological experiments suggested that the strengthened excitation depends on elevated PKC activity. Additionally, postsynaptic AMPA receptor conductance was enhanced and displayed diminished ability to induce plasticity (long-term depression), but this was not dependent upon increased AMPA receptor expression. Morphologically detailed biophysical modeling predicted that synaptic changes, in combination with altered dendritic morphology and intrinsic hypersensitivity, produce increased spontaneous firing rates and a steeper input-output relationship in 3xTg-AD neurons. The results argue against a uniform decrease in synaptic connectivity across the brain in AD, and sheds light on the involvement of deep brain circuits. AD pathology is therefore associated with increased sensitivity of single dopamine neurons, which may help to maintain phasic dopamine signaling in early stages of degeneration.

Autophagy is essential for neuronal homeostasis, while defects in autophagy are implicated in Parkinson disease (PD), a prevalent and progressive neurodegenerative disorder. We used unbiased proteomics to compare cargos degraded by basal autophagy in the brain from two mouse models of PD, PINK1-/- and LRRK2G2019S mice. We find evidence for the upregulation of adaptive pathways to support homeostasis in both PD models. In PINK1-/- mice, we observed increased expression of the selective receptor BNIP3 along with evidence of engagement of other alternative pathways for mitophagy. Despite these changes, we find the rate of autophagic flux in PINK1-/- neurons is decreased. In LRRK2G2019S mice, hyperactive kinase activity known to impair autophagosomal and lysosomal function results in increased secretion of extracellular vesicles and autophagy cargo. In support of this observation, we find reduced levels of PIKFYVE, a negative regulator of extracellular vesicle secretion, in both brain and cortical neurons from LRRK2G2019S mice. Thus, distinct adaptive pathways are activated to compensate for perturbations induced by either loss of PINK1 or hyperactivation of LRRK2. Our findings highlight the engagement of compensatory pathways to maintain homeostasis in the brain, and provide insights into the vulnerabilities these compensatory changes may introduce that may further contribute to PD progression.

The G4C2 hexanucleotide repeat expansion in C9ORF72 is the major genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Despite considerable efforts, the development of mouse models of C9-ALS/FTD useful for therapeutic development has proven challenging due to the intricate interplay of genetic and molecular factors underlying this neurodegenerative disorder, in addition to species differences. This study presents a robust investigation of the cellular pathophysiology and behavioral outcomes in a previously described AAV mouse model of C9-ALS expressing 66 G4C2 hexanucleotide repeats. Despite displaying key molecular ALS pathological markers including RNA foci, dipeptide repeat (DPR) protein aggregation, p62 positive stress granule formation as well as mild gliosis, the AAV-(G4C2)66 mouse model in this study exhibits negligible neuronal loss, no motor deficits, and functionally unimpaired TAR DNA-binding protein-43 (TDP-43). While our findings indicate and support that this is a robust and pharmacologically tractable model for investigating the molecular mechanisms and cellular consequences of (G4C2) repeat driven DPR pathology, it is not suitable for investigating the development of disease associated neurodegeneration, TDP-43 dysfunction, gliosis, and motor performance. Our findings underscore the complexity of ALS pathogenesis involving genetic mutations and protein dysregulation and highlight the need for more comprehensive model systems that reliably replicate the multifaceted cellular and behavioral aspects of C9-ALS.

Spinocerebellar ataxia type 1 (SCA1) and Huntingtons disease (HD), are motor diseases caused by CAG expansions in ATXN1 and HTT, where SCA1 shows prominent cerebellar neurodegeneration and HD shows prominent striatal neurodegeneration, particularly in the Medium Spiny Neurons (MSNs). Since human and mouse studies demonstrate progressive striatal vulnerability in SCA1, we examined age-dependent molecular, cellular and functional striatal attributes in SCA1 (f-ATXN1146Q/2Q) knockin mice, by assessing RNA-sequencing, immunohistochemistry and electrophysiology. Striatal mRNAs are downregulated in SCA1 mice, many in common with HD mice, and specificity in MSNs is supported by the rescue of transcriptomic dysregulation with deletion of mutant Ataxin1 from MSNs. Immunohistochemistry assessed dopamine receptor 1 (D1R) and 2 (D2R) expression in indirect and direct MSNs. In HD mice (HttQ175/Q7), expression of both D1R and D2R proteins in MSNs decreased with age in parallel with their RNA levels. In the SCA1 mouse striatum, D1R protein expression decreased with age as seen in murine HD striatum. In contrast, while D2R protein level was decreased similar to D1R protein at 5-weeks of age, by 40-weeks expression of D2R protein recovered to levels recorded in WT mice. Electrophysiological assessment showed a reduction of excitatory synaptic transmission in SCA1 mouse MSNs, indicating functional deficits early in disease. In contrast to cerebellar and many other aspects of SCA1 pathology known to depend on proper nuclear localization of ATXN1 with an expanded polyglutamine, mutating ATXN1s nuclear localization failed to correct striatal MSN RNA and protein downregulations, indicating a difference in how ATXN1 exerts its pathological effects between the cerebellum and the striatum. Together, these data provide a molecular and cellular basis of striatal pathology in SCA1.

ALS, Amyotrophic lateral sclerosis, a devastating neurodegenerative disease (ND) with no cure, is often caused by abnormal cytosolic aggregation of RNA-binding proteins, the most well-known of which are TDP-43 and FUS. The proteasome is considered one of the major systems that degrades misfolded, including ND-associated, proteins, thereby acting to reduce aggregation, while inhibition of the proteasome increases aggregation. Unexpectedly, we found that proteasome inhibitor treatment significantly reduced ALS-associated mutant FUS aggregation in cells and in primary neurons. This is in sharp contrast to most other ND-associated aggregating proteins, including Huntingtin and TDP-43, for which proteasome inhibitors enhanced aggregation. We further found that this inhibitory effect is dependent on the transcription factor HSF1, suggesting that the underlying mechanism of this effect is transcriptionally-mediated. Since heat shock treatment did not show any effect on FUS aggregation, we hypothesized that proteasome inhibitors elicit a transcriptional program distinct of that of heat shock, which is protective of FUS aggregation. We identified BAG3, a co-chaperone that cooperates with HSP70 in reducing FUS aggregation, as a significant mediator of this effect. We therefore propose BBB-permeable proteasome inhibitors as a potential therapy specific to ALS-FUS.

Altered interaction between striatonigral dopaminergic (DA) inputs and local acetylcholine (ACh) in striatum has long been hypothesized to play a central role in dystonia pathophysiology. Indeed, previous research across various genetic mouse models of human isolated dystonia has identified as a shared endophenotype with paradoxical excitation of striatal cholinergic interneurons (ChIs) activity in response to activation of dopamine D2 receptor (D2R). These mouse models lack a dystonic motor phenotype, which leaves a critical gap in comprehending the role of ACh transmission in the manifestation of dystonia. To tackle this question, we used a combination of ex vivo slice physiology and in vivo monitoring of striatal ACh dynamics in the inducible, phenotypically penetrant, transgenic mouse model of paroxysmal non-kinesigenic dyskinesia (PNKD). We found that, similarly to other genetic models, the PNKD mouse displays D2R-induced paradoxical excitation of ChI firing in ex vivo striatal brain slices. In vivo, caffeine triggers dystonic symptoms while reversing the D2R-mediated excitation of ChIs and desynchronizing the striatal cholinergic network. In WT littermate controls, caffeine stimulates spontaneous locomotion through a similar but reversed mechanism involving an excitatory switch of the D2R control of ChI activity, associated with enhanced cholinergic network synchronization. Together these observations suggest that D2Rs may play an important role in synchronizing the ChI network during heightened movement states. The "paradoxical excitation" described in dystonia models could represent a compensatory or protective mechanism that prevents manifestation of movement abnormalities and allows for phenotypic dystonia when lost.

Pre-symptomatic studies in mouse models of the neurodegenerative motor neuron (MN) disease, Amyotrophic Lateral Sclerosis (ALS) highlight early alterations in intrinsic and synaptic excitability and have supported an excitotoxic theory of MN death. However, a role for synaptic inhibition in disease development is not sufficiently explored among other mechanisms. Since inhibition plays a role in both regulating motor output and in neuroprotection, we examined the age-dependent anatomical changes in inhibitory presynaptic terminals on MN cell bodies using fluorescent immunohistochemistry for GAD67 (GABA) and GlyT2 (glycine) presynaptic proteins comparing ALS-vulnerable trigeminal jaw closer (JC) motor pools with the ALS-resistant extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Our results indicate differential patterns of temporal changes of these terminals in vulnerable versus resilient MNs and relative differences between SOD1G93A and wild-type (WT) MNs. Notably, we found pre-symptomatic up-regulation in inhibitory terminals in the EO MNs while the vulnerable JC MNs mostly showed a decrease in inhibitory terminals. Specifically, there was a statistically significant decrease in the GAD67 somatic abuttal in the SOD1G93A JC MNs compared to WT around P12. Using in vitro patch-clamp electrophysiology, we found a parallel decrease in the ambient GABA-dependent tonic inhibition in the SOD1G93A JC MNs. While it is unclear if the two mechanisms are directly related, pharmacological blockade of specific subtype of GABAA-5 receptors suggests that tonic inhibition can control MN recruitment threshold. Furthermore, reduction in tonic GABA current as observed here in the mutant, identifies a putative molecular mechanism explaining our observations of hyperexcitable shifts in JC MN recruitment threshold in the SOD1G93A mouse. Lastly, we showcase non-parametric resampling-based bootstrap statistics for data analyses, and provide the Python code on GitHub for wider reuse.