**4. Evidence that the cerebellum is involved in ASD**

In the recent years, there has been increasing evidence showing a crucial role for the cerebellum in the etiology of ASD [38–40]. Although the field of cerebellar research in disorders of social cognition such as autism is still in its early stages, below we will describe the main structural and functional cerebellar abnormalities that have been described to date in autism, which provide strong evidence to grant further research.

### **4.1 Structural cerebellar abnormalities found in autism**

The cerebellum is actually one of the most consistent sites of neural abnormalities found in autism [41]. Specifically, the reduced size and number of PCs are among the most replicated findings in postmortem brain tissue of individuals with autism [19]. This reduction in PC in autism patients has been found to be more pronounced in the Crus I/Crus II region of lobule VIIA [42]. Accordingly, a reduction in gray matter volume, smaller ratio of gray to white matter, and smaller vermis lobules VI–VII have been found in children with autism compared to controls [43, 44]. Further, in ASD patients, the degree of reduction in gray matter of Crus I/ Crus II has been repeatedly found to correlate with the severity of symptoms in the social interaction and communication behavioral domains of ASD [39, 45]. Of note, some reports using adult brains indicate the presence of gliosis as an accompanying factor to the reduction of PC [46]. Other observed cerebellar cellular abnormalities are the presence of neuro-inflammatory processes [47].

Besides structural and cellular alterations, molecular abnormalities have also been reported in the cerebellum of ASD individuals. Alterations in the distribution of the mRNA levels of glutamic acid decarboxylase 67 (GAD67), an enzyme involved in the synthesis of the inhibitory neurotransmitter GABA, have been found. Thus, decreased GAD67 mRNA has been reported in PC [48], while increased GAD67 mRNA has been reported in cerebellar interneurons [49]. These cerebellar imbalances could account for the proposed E/I disequilibrium in ASD, as they could affect cerebro-cerebellar circuits [50].

#### **4.2 Abnormalities of cerebellar function**

Studies in humans using resting-state functional connectivity (rsFC) techniques have reported connectivity alterations between cerebellar and cortical areas in

autism compared to typically developing individuals. Overall, a general cerebrocerebellar over-connectivity has been found in the ASD group. However, both hyper-connectivity and hypo-connectivity have been reported depending on the regions analyzed. For example, cerebellar-sensorimotor FC (premotor and primary motor cortices, somatosensory temporal cortex, and occipital lobe) has been found to be atypically increased in ASD, while cerebellar-supramodal FC (prefrontal cortex, posterior parietal cortex, and inferior and middle temporal gyri) has been found to be decreased [51]. Analysis of cerebellar FC with language-related areas revealed a significantly reduced FC in ASD between the cerebellum and Broca's area and Wernicke's area [52], suggesting that the cerebellum plays a role in language functioning. Studies aiming at assessing the developmental pattern of cerebellocerebral FC also report developmental alterations in ASD. FC between the cerebellum and subcortical regions was found to decrease in neurotypical individuals, while it increased in ASD [53]. It must be noted that no specific correlation between FC patterns and autism behavior has been detected, although reduced connectivity seems to be accompanied by an increase in the severity of the disorder [52], as assessed by the Social Communication Questionnaire, an ASD screening measure consisting of a brief (40-item) parent report that focuses on ASD symptomatology likely to be observed by a primary caregiver.

Few studies have investigated to date the FC between the cerebellum and cortical areas during task performance in ASD. During a sequential finger-tapping task, activations in motor circuits were found in both cases and controls. However, children with typical development showed activation of cerebellar structures that were silent in autistic children (lobules IV/V and anterior cerebellum). In addition, a reduced FC between premotor areas and the cerebellum was observed in autistic children, suggesting alterations in long-range cerebro-cerebellar connections [54]. In a task that requires perception and imitation of human actions, fMRI detected an engagement between the posterior superior temporal sulcus (pSTS) and the cerebellar region Crus I [55]. Interestingly, the degree of functional coactivation of pSTS and Crus I could predict social deficits in ASD in the "mentalizing skills" questionnaire, a parent report for specific social cognition skills based on imaginative mental activity that allows an understanding of the behavior of other people (intentions, needs, desires, or goals). Thus, stronger Crus I-pSTS interactions were associated with better mentalizing ability [55]. On a similar note, during a task that involves decoding the interactions between animated figures, aimed at examining the "theory of mind" network, that is, the ability to attribute mental states to others, a reduced cerebellar activation, particularly in Crus I, in participants with ASD was found [56]. Although many more studies are needed, overall the abovepresented data indicate a role for cerebellar connections with key cortical social brain sites and, specifically for region Crus I/Crus II, in the pathogenesis of ASD.
